RESUMO
Free extracellular heme has been shown to activate several compartments of innate immunity, acting as a danger-associated molecular pattern (DAMP) in hemolytic diseases. Although localized endothelial barrier (EB) disruption is an important part of inflammation that allows circulating leukocytes to reach inflamed tissues, non-localized/deregulated disruption of the EB can lead to widespread microvascular hyperpermeability and secondary tissue damage. In mouse models of sickle cell disease (SCD), EB disruption has been associated with the development of a form of acute lung injury that closely resembles acute chest syndrome (ACS), and that can be elicited by acute heme infusion. Here we explored the effect of heme on EB integrity using human endothelial cell monolayers, in experimental conditions that include elements that more closely resemble in vivo conditions. EB integrity was assessed by electric cell-substrate impedance sensing in the presence of varying concentrations of heme and sera from SCD patients or healthy volunteers. Heme caused a dose-dependent decrease of the electrical resistance of cell monolayers, consistent with EB disruption, which was confirmed by staining of junction protein VE-cadherin. In addition, sera from SCD patients, but not from healthy volunteers, were also capable to induce EB disruption. Interestingly, these effects were not associated with total heme levels in serum. However, when heme was added to sera from SCD patients, but not from healthy volunteers, EB disruption could be elicited, and this effect was associated with hemopexin serum levels. Together our in vitro studies provide additional support to the concept of heme as a DAMP in hemolytic conditions.
Assuntos
Anemia Falciforme/imunologia , Antígenos CD/imunologia , Caderinas/imunologia , Heme/imunologia , Hemopexina/imunologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Anemia Falciforme/sangue , Antígenos CD/metabolismo , Caderinas/metabolismo , Heme/metabolismo , Hemopexina/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/patologia , HumanosRESUMO
Compelling evidence suggests that fibroblast growth factor 2 (FGF2), overexpressed in melanomas, plays an important role in tumor growth, angiogenesis and metastasis. In this study, we evaluated the therapeutic use of a new anti-FGF2 monoclonal antibody (mAb), 3F12E7, using for that the B16-F10 melanoma model. The FGF2 neutralizing effect of this antibody was certified by in vitro assays, which allowed the further track of its possible in vivo application. 3F12E7 mAb could be retained in B16-F10 tumors, as shown by antibody low-pH elution and nuclear medicine studies, and also led to reduction in number and size of metastatic foci in lungs, when treatment starts one day after intravenous injection of B16-F10 cells. Such data were accompanied by decreased CD34(+) tumor vascular density and impaired subcutaneous tumor outgrowth. Treatments starting one week after melanoma cell intravenous injection did not reduce tumor burden, remaining the therapeutic effectiveness restricted to early-adopted regimens. Altogether, the presented anti-FGF2 3F12E7 mAb stands as a promising agent to treat metastatic melanoma tumors in adjuvant settings.
Assuntos
Inibidores da Angiogênese/farmacologia , Anticorpos Monoclonais/farmacologia , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Neoplasias Pulmonares/prevenção & controle , Melanoma Experimental/tratamento farmacológico , Neovascularização Patológica , Neovascularização Fisiológica/efeitos dos fármacos , Inibidores da Angiogênese/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Antígenos CD34/metabolismo , Linhagem Celular Tumoral , Fator 2 de Crescimento de Fibroblastos/imunologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Imuno-Histoquímica , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Masculino , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/metabolismo , Melanoma Experimental/secundário , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Tomografia Computadorizada de Emissão de Fóton Único , Tomografia Computadorizada por Raios X , Carga TumoralRESUMO
The hepatitis C virus (HCV) encodes approximately 10 different structural and non-structural proteins, including the envelope glycoprotein 2 (E2). HCV proteins, especially the envelope proteins, bind to cell receptors and can damage tissues. Endothelial inflammation is the most important determinant of fibrosis progression and, consequently, cirrhosis. The aim of this study was to evaluate and compare the inflammatory response of endothelial cells to two recombinant forms of the HCV E2 protein produced in different expression systems (Escherichia coli and Pichia pastoris). We observed the induction of cell death and the production of nitric oxide, hydrogen peroxide, interleukin-8 and vascular endothelial growth factor A in human umbilical vein endothelial cells (HUVECs) stimulated by the two recombinant E2 proteins. The E2-induced apoptosis of HUVECs was confirmed using the molecular marker PARP. The apoptosis rescue observed when the antioxidant N-acetylcysteine was used suggests that reactive oxygen species are involved in E2-induced apoptosis. We propose that these proteins are involved in the chronic inflammation caused by HCV.
Assuntos
Hepacivirus/metabolismo , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/patologia , Óxido Nítrico/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas do Envelope Viral/metabolismo , Apoptose/genética , Arginase/metabolismo , Sobrevivência Celular , Escherichia coli/metabolismo , Fibrose , Expressão Gênica/genética , Engenharia Genética/métodos , Vetores Genéticos/metabolismo , Hepacivirus/imunologia , Antígenos da Hepatite C/metabolismo , Humanos , Inflamação/metabolismo , Interleucina-8/metabolismo , Pichia/metabolismo , Plasmídeos/metabolismo , Proteínas Recombinantes , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The hepatitis C virus (HCV) encodes approximately 10 different structural and non-structural proteins, including the envelope glycoprotein 2 (E2). HCV proteins, especially the envelope proteins, bind to cell receptors and can damage tissues. Endothelial inflammation is the most important determinant of fibrosis progression and, consequently, cirrhosis. The aim of this study was to evaluate and compare the inflammatory response of endothelial cells to two recombinant forms of the HCV E2 protein produced in different expression systems (Escherichia coli and Pichia pastoris). We observed the induction of cell death and the production of nitric oxide, hydrogen peroxide, interleukin-8 and vascular endothelial growth factor A in human umbilical vein endothelial cells (HUVECs) stimulated by the two recombinant E2 proteins. The E2-induced apoptosis of HUVECs was confirmed using the molecular marker PARP. The apoptosis rescue observed when the antioxidant N-acetylcysteine was used suggests that reactive oxygen species are involved in E2-induced apoptosis. We propose that these proteins are involved in the chronic inflammation caused by HCV.
Assuntos
Humanos , Hepacivirus/metabolismo , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/patologia , Óxido Nítrico/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteínas do Envelope Viral/metabolismo , Apoptose/genética , Arginase/metabolismo , Sobrevivência Celular , Escherichia coli/metabolismo , Fibrose , Expressão Gênica/genética , Engenharia Genética/métodos , Vetores Genéticos/metabolismo , Hepacivirus/imunologia , Antígenos da Hepatite C/metabolismo , Inflamação/metabolismo , /metabolismo , Pichia/metabolismo , Plasmídeos/metabolismo , Proteínas Recombinantes , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
INTRODUCTION: In patients, a transient decrease in peripheral blood lymphocyte counts was observed following intraperitoneal administration of the trifunctional monoclonal antibody catumaxomab (anti-human EpCAM x anti-human CD3). The aim of this study was to clarify the observed effect in a preclinical mouse model and to analyse the related mechanism of action in vitro. MATERIALS AND METHODS: A related antibody, BiLu (antihuman EpCAM x anti-mouse CD3), was administered to mice and blood leukocytes were analysed. In vitro studies measured activation and cytokine secretion from human peripheral blood mononuclear cells (PBMC). For the analysis of T cell adhesion, PBMC were preincubated with catumaxomab and then co-cultured with human endothelial cells (HUVEC); T cell adhesion was assessed in the presence or absence of endothelial cell preactivation by TNFα. Adherent T cells were determined by flow cytometry. RESULTS: Treatment of mice with BiLu resulted in a dosedependent transient decrease in CD3+ T cells (both CD4+ and CD8+) that returned to the normal range within 48 h. Catumaxomab physiologically activated T cells in vitro (increased CD69 expression) and induced cytokine release (TNFα, IFNγ). TNFα increased expression of adhesion molecules CD54 and CD62E on endothelial cells. Furthermore, catumaxomab dose-dependently enhanced adhesion of T cells to endothelial cells. Adhesion was further increased when endothelial cells were preactivated with TNFα. CONCLUSIONS: Catumaxomab increases adhesion of T cells to endothelial cells due to antibody-mediated activation of T cells and production of T cell cytokines that up-regulate endothelial cell adhesion molecules. These results provide a mechanistic rationale for the transient, reversible decrease in lymphocyte counts observed following catumaxomab administration in patients, which is likely to be due to redistribution of lymphocytes.
Assuntos
Anticorpos Biespecíficos/farmacologia , Adesão Celular/imunologia , Movimento Celular/imunologia , Células Endoteliais da Veia Umbilical Humana/citologia , Leucócitos Mononucleares/citologia , Linfócitos T/citologia , Animais , Antígenos de Neoplasias/imunologia , Complexo CD3/imunologia , Moléculas de Adesão Celular/imunologia , Células Cultivadas , Citocinas/metabolismo , Molécula de Adesão da Célula Epitelial , Feminino , Citometria de Fluxo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/imunologia , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Vaccination with xenogeneic and syngeneic endothelial cells is effective for inhibiting tumor growth. Nontoxic diphtheria toxin (CRM197), as an immunogen or as a specific inhibitor of heparin-binding EGF-like growth factor, has shown promising antitumor activity. Therefore, immunization with or administration of viable human umbilical vein endothelial cells (HUVECs) combined with CRM197 could have an enhanced antitumor effect. Six-week-old C57BL/6J male mice were vaccinated with viable HUVECs, 1 x 10(6) viable HUVECs combined with 100 μg CRM197, or 100 μg CRM197 alone by ip injections once a week for 4 consecutive weeks. RM-1 cells (5 x 10(5)) were inoculated by sc injection as a preventive procedure. During the therapeutic procedure, 6-week-old male C57BL/6J mice were challenged with 1 x 10(5) RM-1 cells, then injected sc with 1 x 10(6) viable HUVECs, 1 x 10(6) viable HUVECs + 100 μg CRM197, and 100 μg CRM197 alone twice a week for 4 consecutive weeks. Tumor volume and life span were monitored. We also investigated the effects of immunization with HUVECs on the aortic arch wall and on wound healing. Vaccination with or administration of viable HUVECs+CRM197 enhanced the inhibition of RM-1 prostatic carcinoma by 24 and 29 percent, respectively, and prolonged the life span for 3 and 4 days, respectively, compared with those of only vaccination or administration with viable HUVECs of tumor-bearing C57BL/6J mice. Furthermore, HUVEC immunization caused some damage to the aortic arch wall but did not have remarkable effects on the rate of wound healing; the wounds healed in approximately 13 days. Treatment with CRM197 in combination with viable HUVECs resulted in a marked enhancement of the antitumor effect in the preventive or therapeutic treatment for prostatic carcinoma in vivo, suggesting a novel combination for anti-cancer therapy.
Assuntos
Animais , Humanos , Masculino , Camundongos , Proteínas de Bactérias/uso terapêutico , Células Endoteliais da Veia Umbilical Humana/transplante , Neoplasias da Próstata/terapia , Proteínas de Bactérias/imunologia , Terapia Combinada/métodos , Células Endoteliais da Veia Umbilical Humana/imunologia , Neoplasias da Próstata/imunologia , Transplante Heterólogo , Transplante Isogênico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Vaccination with xenogeneic and syngeneic endothelial cells is effective for inhibiting tumor growth. Nontoxic diphtheria toxin (CRM197), as an immunogen or as a speciï¬c inhibitor of heparin-binding EGF-like growth factor, has shown promising antitumor activity. Therefore, immunization with or administration of viable human umbilical vein endothelial cells (HUVECs) combined with CRM197 could have an enhanced antitumor effect. Six-week-old C57BL/6J male mice were vaccinated with viable HUVECs, 1 x 10(6) viable HUVECs combined with 100 µg CRM197, or 100 µg CRM197 alone by ip injections once a week for 4 consecutive weeks. RM-1 cells (5 x 10(5)) were inoculated by sc injection as a preventive procedure. During the therapeutic procedure, 6-week-old male C57BL/6J mice were challenged with 1 x 10(5) RM-1 cells, then injected sc with 1 x 10(6) viable HUVECs, 1 x 10(6) viable HUVECs + 100 µg CRM197, and 100 µg CRM197 alone twice a week for 4 consecutive weeks. Tumor volume and life span were monitored. We also investigated the effects of immunization with HUVECs on the aortic arch wall and on wound healing. Vaccination with or administration of viable HUVECs+CRM197 enhanced the inhibition of RM-1 prostatic carcinoma by 24 and 29%, respectively, and prolonged the life span for 3 and 4 days, respectively, compared with those of only vaccination or administration with viable HUVECs of tumor-bearing C57BL/6J mice. Furthermore, HUVEC immunization caused some damage to the aortic arch wall but did not have remarkable effects on the rate of wound healing; the wounds healed in approximately 13 days. Treatment with CRM197 in combination with viable HUVECs resulted in a marked enhancement of the antitumor effect in the preventive or therapeutic treatment for prostatic carcinoma in vivo, suggesting a novel combination for anti-cancer therapy.