RESUMO
The classic pathway (CP) of complement is believed to significantly contribute to alloantibody-mediated transplant injury, and targeted complement inhibition is currently considered to be a promising approach for preventing rejection. Here, we investigated the mode of action and efficacy of the humanized anti-C1s monoclonal antibody TNT009 and its parental mouse variant, TNT003, in preclinical in vitro models of HLA antibody-triggered CP activation. In flow cytometric assays, we measured the attachment of C1 subcomponents and C4/C3 split products (C4b/d, C3b/d) to HLA antigen-coated flow beads or HLA-mismatched aortic endothelial cells and splenic lymphocytes. Anti-C1s antibodies profoundly inhibited C3 activation at concentrations >20 µg/mL, in both solid phase and cellular assays. While C4 activation was also prevented, this was not the case for C1 subcomponent attachment. Analysis of serum samples obtained from 68 sensitized transplant candidates revealed that the potency of inhibition was related to the extent of baseline CP activation. This study demonstrates that anti-C1s antibodies TNT009 and TNT003 are highly effective in blocking HLA antibody-triggered complement activation downstream of C1. Our results provide the foundation for clinical studies designed to investigate the potential of TNT009 in the treatment or prevention of complement-mediated tissue injury in sensitized transplant recipients.
Assuntos
Anticorpos Monoclonais/farmacologia , Ativação do Complemento/imunologia , Complemento C1s/imunologia , Rejeição de Enxerto/tratamento farmacológico , Antígenos HLA/imunologia , Isoanticorpos/efeitos adversos , Transplante de Rim/efeitos adversos , Animais , Seguimentos , Taxa de Filtração Glomerular , Rejeição de Enxerto/etiologia , Sobrevivência de Enxerto , Humanos , Falência Renal Crônica/imunologia , Falência Renal Crônica/cirurgia , Testes de Função Renal , Camundongos , PrognósticoRESUMO
BACKGROUND: As metastasis is the prime cause of death from malignancies, there is vibrant interest to discover options for the management of the different mechanistic steps of tumour spreading. Some approved pharmaceuticals exhibit activities against diseases they have not been developed for. In order to discover such activities that might attenuate lymph node metastasis, we investigated 225 drugs, which are approved by the US Food and Drug Administration. METHODS: A three-dimensional cell co-culture assay was utilised measuring tumour cell-induced disintegrations of the lymphendothelial wall through which tumour emboli can intravasate as a limiting step in lymph node metastasis of ductal breast cancer. The disintegrated areas in the lymphendothelial cell (LEC) monolayers were induced by 12(S)-HETE, which is secreted by MCF-7 tumour cell spheroids, and are called 'circular chemorepellent induced defects' (CCIDs). The putative mechanisms by which active drugs prevented the formation of entry gates were investigated by western blotting, NF-κB activity assay and by the determination of 12(S)-HETE synthesis. RESULTS: Acetohexamide, nifedipin, isoxsuprine and proadifen dose dependently inhibited the formation of CCIDs in LEC monolayers and inhibited markers of epithelial-to-mesenchymal-transition and migration. The migration of LECs is a prerequisite of CCID formation, and these drugs either repressed paxillin levels or the activities of myosin light chain 2, or myosin-binding subunit of myosin phosphatase. Isoxsuprine inhibited all three migration markers, and isoxsuprine and acetohexamide suppressed the synthesis of 12(S)-HETE, whereas proadifen and nifedipin inhibited NF-κB activation. Both the signalling pathways independently cause CCID formation. CONCLUSION: The targeting of different mechanisms was most likely the reason for synergistic effects of different drug combinations on the inhibition of CCID formation. Furthermore, the treatment with drug combinations allowed also a several-fold reduction in drug concentrations. These results encourage further screening of approved drugs and their in vivo testing.
Assuntos
Acetoexamida/farmacologia , Neoplasias da Mama/tratamento farmacológico , Endotélio Linfático/efeitos dos fármacos , Isoxsuprina/farmacologia , Vasos Linfáticos/efeitos dos fármacos , Nifedipino/farmacologia , Proadifeno/farmacologia , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/tratamento farmacológico , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Adesão Celular/efeitos dos fármacos , Movimento Celular , Quimiotaxia/efeitos dos fármacos , Técnicas de Cocultura , Sinergismo Farmacológico , Endotélio Linfático/citologia , Endotélio Linfático/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Hipoglicemiantes/farmacologia , Metástase Linfática , Vasos Linfáticos/irrigação sanguínea , Vasos Linfáticos/patologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares/metabolismo , Células Tumorais Cultivadas , Vasodilatadores/farmacologiaRESUMO
BACKGROUND: Many cancers spread through lymphatic routes, and mechanistic insights of tumour intravasation into the lymphatic vasculature and targets for intervention are limited. The major emphasis of research focuses currently on the molecular biology of tumour cells, while still little is known regarding the contribution of lymphatics. METHODS: Breast cancer cell spheroids attached to lymphendothelial cell (LEC) monolayers were used to investigate the process of intravasation by measuring the areas of 'circular chemorepellent-induced defects' (CCID), which can be considered as entry gates for bulky tumour intravasation. Aspects of tumour cell intravasation were furthermore studied by adhesion assay, and siRNA-mediated knockdown of intracellular adhesion molecule-1 (ICAM-1). Replacing cancer spheroids with the CCID-triggering compound 12(S)-hydroxyeicosatetraenoic acid (HETE) facilitated western blot analyses of Bay11-7082- and baicalein-treated LECs. RESULTS: Binding of LECs to MCF-7 spheroids, which is a prerequisite for CCID formation, was mediated by ICAM-1 expression, and this depended on NF-κB and correlated with the expression of the prometastatic factor S100A4. Simultaneous inhibition of NF-κB with Bay11-7082 and of arachidonate lipoxygenase (ALOX)-15 with baicalein prevented CCID formation additively. CONCLUSION: Two mechanisms contribute to CCID formation: ALOX15 via the generation of 12(S)-HETE by MCF-7 cells, which induces directional migration of LECs, and ICAM-1 in LECs under control of NF-κB, which facilitates adhesion of MCF-7 cells to LECs.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Adesão Celular/efeitos dos fármacos , Endotélio Linfático/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/química , NF-kappa B/antagonistas & inibidores , Nitrilas/farmacologia , Esferoides Celulares/efeitos dos fármacos , Sulfonas/farmacologia , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Movimento Celular , Quimiotaxia/efeitos dos fármacos , Endotélio Linfático/citologia , Endotélio Linfático/metabolismo , Feminino , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
BACKGROUND: The intravasation of breast cancer into the lymphendothelium is an early step of metastasis. Little is known about the mechanisms of bulky cancer invasion into lymph ducts. METHODS: To particularly address this issue, we developed a 3-dimensional co-culture model involving MCF-7 breast cancer cell spheroids and telomerase-immortalised human lymphendothelial cell (LEC) monolayers, which resembles intravasation in vivo and correlated the malignant phenotype with specific protein expression of LECs. RESULTS: We show that tumour spheroids generate 'circular chemorepellent-induced defects' (CCID) in LEC monolayers through retraction of LECs, which was induced by 12(S)-hydroxyeicosatetraenoic acid (HETE) secreted by MCF-7 spheroids. This 12(S)-HETE-regulated retraction of LECs during intravasation particularly allowed us to investigate the key regulators involved in the motility and plasticity of LECs. In all, 12(S)-HETE induced pro-metastatic protein expression patterns and showed NF-κB-dependent up-regulation of the mesenchymal marker protein S100A4 and of transcriptional repressor ZEB1 concomittant with down-regulation of the endothelial adherence junction component VE-cadherin. This was in accordance with â¼50% attenuation of CCID formation by treatment of cells with 10 µM Bay11-7082. Notably, 12(S)-HETE-induced VE-cadherin repression was regulated by either NF-κB or by ZEB1 since ZEB1 siRNA knockdown abrogated not only 12(S)-HETE-mediated VE-cadherin repression but inhibited VE-cadherin expression in general. INTERPRETATION: These data suggest an endothelial to mesenchymal transition-like process of LECs, which induces single cell motility during endothelial transmigration of breast carcinoma cells. In conclusion, this study demonstrates that the 12(S)-HETE-induced intravasation of MCF-7 spheroids through LECs require an NF-κB-dependent process of LECs triggering the disintegration of cell-cell contacts, migration, and the generation of CCID.
Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/farmacologia , Neoplasias da Mama/patologia , Carcinoma/patologia , Transdiferenciação Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , NF-kappa B/fisiologia , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Carcinoma/metabolismo , Linhagem Celular Transformada , Movimento Celular/efeitos dos fármacos , Técnicas de Cocultura , Células Endoteliais/fisiologia , Feminino , Humanos , Mesoderma/efeitos dos fármacos , Mesoderma/fisiologia , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Invasividade Neoplásica , Nitrilas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sulfonas/farmacologia , Células Tumorais CultivadasRESUMO
BACKGROUND: Digalloyl-resveratrol (di-GA) is a synthetic compound aimed to combine the biological effects of the plant polyhydroxy phenols gallic acid and resveratrol, which are both radical scavengers and cyclooxygenase inhibitors exhibiting anticancer activity. Their broad spectrum of activities may probably be due to adjacent free hydroxyl groups. METHODS: Protein activation and expression were analysed by western blotting, deoxyribonucleoside triphosphate levels by HPLC, ribonucleotide reductase activity by (14)C-cytidine incorporation into nascent DNA and cell-cycle distribution by FACS. Apoptosis was measured by Hoechst 33258/propidium iodide double staining of nuclear chromatin and the formation of gaps into the lymphendothelial barrier in a three-dimensional co-culture model consisting of MCF-7 tumour cell spheroids and human lymphendothelial monolayers. RESULTS: In HL-60 leukaemia cells, di-GA activated caspase 3 and dose-dependently induced apoptosis. It further inhibited cell-cycle progression in the G1 phase by four different mechanisms: rapid downregulation of cyclin D1, induction of Chk2 with simultaneous downregulation of Cdc25A, induction of the Cdk-inhibitor p21(Cip/Waf) and inhibition of ribonucleotide reductase activity resulting in reduced dCTP and dTTP levels. Furthermore, di-GA inhibited the generation of lymphendothelial gaps by cancer cell spheroid-secreted lipoxygenase metabolites. Lymphendothelial gaps, adjacent to tumour bulks, can be considered as gates facilitating metastatic spread. CONCLUSION: These data show that di-GA exhibits three distinct anticancer activities: induction of apoptosis, cell-cycle arrest and disruption of cancer cell-induced lymphendothelial disintegration.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Ácido Gálico/análogos & derivados , Células HL-60/efeitos dos fármacos , Estilbenos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Corantes , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Citometria de Fluxo , Ácido Gálico/farmacologia , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/fisiologia , Células HL-60/citologia , Humanos , Pulmão/citologia , Pulmão/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Humoral alloreactivity is well established to predict adverse allograft outcomes. However, in some recipients, alloantibodies may also occur in the absence of graft dysfunction. We evaluated if and how often complement- and noncomplement-fixing alloantibodies are detectable in stable recipients and whether, in this context, they affect long-term outcomes. Sera obtained from 164 kidney transplant recipients at 2, 6 and 12 months were evaluated by FlowPRA screening and single-antigen testing for detection of IgG- or C4d-fixing HLA panel reactivity and donor-specific antibodies (DSA). Applying stringent criteria, we selected 34 patients with an uneventful 1-year course (no graft dysfunction or rejection) and excellent graft function at 12 months [estimated glomerular filtration rate (eGFR) >or=60 mL/min and proteinuria Assuntos
Anticorpos/sangue
, Antígenos HLA/imunologia
, Transplante de Rim/imunologia
, Avaliação de Resultados em Cuidados de Saúde
, Transplante
, Adulto
, Complemento C4b
, Feminino
, Rejeição de Enxerto/epidemiologia
, Rejeição de Enxerto/imunologia
, Humanos
, Imunoglobulina G/sangue
, Incidência
, Estudos Longitudinais
, Masculino
, Pessoa de Meia-Idade
, Fragmentos de Peptídeos/sangue
, Valor Preditivo dos Testes
, Estudos Retrospectivos
, Transplante Homólogo
RESUMO
Capillary C4d deposition is a valuable marker of antibody-mediated rejection (AMR). In this analysis, flow cytometric detection of alloantibody-triggered C4d deposition to HLA antigen-coated microparticles ([C4d]FlowPRA) was evaluated for its value as a marker for C4d deposition in renal allografts. For comparative analysis, 105 first renal biopsies performed for graft dysfunction and an equal number of concurrent sera were subjected to immunohistochemistry and [C4d] plus standard [IgG]FlowPRA, respectively. C4d deposition/fixation was detected in 17 biopsies and, applying [C4d]FlowPRA HLA class I and II screening, also in a small number of corresponding sera (N = 20). IgG reactivity detected by standard [IgG]FlowPRA was more frequent (49% of sera). Comparing [C4d]FlowPRA screening with capillary C4d staining, we found a high level of specificity (0.92 [95% confidence interval: 0.86-0.98]), which far exceeded that calculated for [IgG]FlowPRA (0.60 [0.50-0.70]). [IgG]FlowPRA screening, however, turned out to be superior in terms of sensitivity (0.94 [0.83-1.05] vs. 0.76 [0.56-0.97] calculated for C4d-fixing panel reactivity). Remarkably, posttransplant single antigen testing for identification of complement-fixing donor-specific alloreactivities failed to improve the predictive value of FlowPRA-based serology. In conclusion, our results suggest that detection of complement-fixing HLA panel reactivity could provide a specific tool for monitoring of C4d-positive AMR.
Assuntos
Complemento C4b/metabolismo , Antígenos HLA/imunologia , Isoanticorpos/análise , Transplante de Rim/imunologia , Rim/irrigação sanguínea , Rim/metabolismo , Fragmentos de Peptídeos/metabolismo , Adulto , Capilares/imunologia , Capilares/metabolismo , Testes de Fixação de Complemento , Feminino , Humanos , Isoanticorpos/fisiologia , Rim/imunologia , Masculino , Pessoa de Meia-Idade , Transplante HomólogoRESUMO
BACKGROUND: Although endothelial cells express insulin receptors, it is controversially discussed whether the endothelium represents an insulin-responsive tissue. Since available data are primarily restricted to animal endothelial cells, this study tested (i) whether insulin affects glucose metabolism in human endothelium; (ii) whether insulin sensitivity is different in micro- versus macrovascular endothelial cells; and (iii) whether glucose concentration in the incubation medium affects the cells' response to insulin. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVECs), human adult saphenous vein endothelial cells (HAVECs), human aortic endothelial cells (HAEC), and human retinal endothelial cells (HRECs) as well as human smooth muscle cells were incubated with/without insulin (0.3 nmol L(-1) or 1 micromol L(-1)). Glucose transport, glycogen synthesis, glycogen content, lactate release, and expression of phospho-Akt, Akt, and endothelial nitric oxide synthase (eNOS) were determined. RESULTS: In HUVECs and HRECs, insulin (1 micromol L(-1)) increased (P < 0.05) eNOS expression by ~70% and doubled Akt phosphorylation, but the latter was by far more pronounced in human smooth muscle cells (+1093 +/- 500%, P < 0.05). In human smooth muscle cells, insulin (1 micromol L(-1)) stimulated glycogen synthesis by 67 +/- 11% (P < 0.01). In human micro- (HRECs) and macrovascular endothelial cells (HUVECs, HAVECs and HAECs), insulin, however, failed to stimulate glucose transport, glycogen synthesis, glycogen content, or lactate release under various conditions, i.e. after glucose deprivation or in medium with normal (5.5 mmol L(-1)) or high glucose (30 mmol L(-1)). CONCLUSIONS: Insulin stimulated glycogen synthesis and Akt phosphorylation in human smooth muscle cells. In human micro- and macrovascular endothelial cells, insulin, however, failed to affect glucose uptake and metabolism under all experimental conditions applied, whereas it increased Akt phosphorylation and eNOS expression.
Assuntos
Células Endoteliais/efeitos dos fármacos , Proteínas Facilitadoras de Transporte de Glucose/efeitos dos fármacos , Glucose/metabolismo , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/metabolismo , Humanos , Miócitos de Músculo Liso/metabolismo , Óxido Nítrico Sintase Tipo III/efeitos dos fármacos , Fosforilação/efeitos dos fármacosRESUMO
Accumulation of inflammatory cells within capillaries is a common morphologic feature of humoral renal allograft rejection and is most easily appreciated if it occurs in glomeruli. The aim of our study was to determine the amount and composition of immune cells within glomeruli and peritubular capillaries (PTC) in cellular and humoral allograft rejection. Immunofluorescent double-labeling for CD31 and CD3 or CD68 was used for phenotyping and enumerating immune cells within glomeruli and PTC. The major findings are: (1) accumulation of immune cells in PTC is far more common than it would be anticipated based on the assessment by conventional histology; (2) it is not the absolute number of immune cells accumulating within capillaries, but rather the composition of the intracapillary cell population that distinguishes humoral rejection from cellular rejection and (3) in C4d positive biopsies a predominantly monocytic cell population accumulates not only within glomeruli but also within PTC. The median value of monocyte/T-cell ratio within PTC was 2.3 in C4d positive biopsies but only 1 (p = 0.0008) in C4d negative biopsies. Given their prominent presence within capillaries and their extensive biological versatility monocytes might contribute to the capillary damage observed in acute and chronic allograft rejection.