RESUMEN
In vitro angiogenesis assays are commonly used to assess pro- or anti-angiogenic drug properties. Extracellular matrix (ECM) substitutes such as Matrigel and collagen gel became very popular in in vitro 3D angiogenesis assays as they enable tubule formation by endothelial cells from culture or aortic rings. However, these assays are usually used with a single cell type, lacking the complex cellular interactions occurring during angiogenesis. Here, we report a novel angiogenesis assay using egg white as ECM substitute. We found that, similar to Matrigel, egg white elicited prevascular network formation by endothelial and/or smooth muscle cell coculture. This matrix was suitable for various cells from human, mouse, and rat origin. It is compatible with aortic ring assay and also enables vascular and tumor cell coculture. Through simple labeling (DAPI, Hoechst 33258), cell location and resulting prevascular network formation can easily be quantified. Cell transfection with green fluorescent protein improved whole cell visualization and 3D structure characterization. Finally, egg-based assay dedicated to angiogenesis studies represents a reliable and cost-effective way to produce and analyze data regarding drug effects on vascular cells.
Asunto(s)
Neovascularización Fisiológica/efectos de los fármacos , Animales , Animales Modificados Genéticamente , Aorta Torácica/citología , Bisbenzimidazol , Técnicas de Cocultivo/métodos , Colágeno , Combinación de Medicamentos , Evaluación Preclínica de Medicamentos/métodos , Clara de Huevo , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Colorantes Fluorescentes , Células Endoteliales de la Vena Umbilical Humana , Humanos , Indoles , Laminina , Ratones , Miocitos del Músculo Liso/citología , Miocitos del Músculo Liso/efectos de los fármacos , Proteoglicanos , Ratas , Especificidad de la EspecieRESUMEN
The genome of a new human polyomavirus, known as Merkel cell polyomavirus (MCV), has recently been reported to be integrated within the cellular DNA of Merkel cell carcinoma (MCC), a rare human skin cancer. To investigate MCV seroprevalence in the general population, we expressed three different MCV VP1 in insect cells using recombinant baculoviruses. Viruslike particles (VLPs) were obtained with only one of the three VP1 genes. High-titer antibodies against VP1 VLPs were detected in mice immunized with MCV VLPs, and limited cross-reactivity was observed with BK polyomavirus (BKV) and lymphotropic polyomavirus (LPV). MCV antibodies were detected in 77% of the general population, with no variations according to age.
Asunto(s)
Anticuerpos Antivirales/sangre , Antígenos Virales , Carcinoma de Células de Merkel/virología , Infecciones por Polyomavirus/diagnóstico , Poliomavirus/inmunología , Virosomas , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antígenos Virales/genética , Antígenos Virales/aislamiento & purificación , Virus BK/inmunología , Baculoviridae/genética , Línea Celular , Reacciones Cruzadas , Femenino , Expresión Génica , Vectores Genéticos , Humanos , Insectos , Masculino , Células de Merkel/virología , Ratones , Microscopía Electrónica de Transmisión , Poliomavirus/genética , Sensibilidad y Especificidad , Virosomas/genética , Virosomas/aislamiento & purificación , Virosomas/ultraestructura , Adulto JovenRESUMEN
The human cytomegalovirus (HCMV) terminase complex is part of DNA-packaging machinery that delivers a unit-length genome into a procapsid. Sequence comparison of herpesvirus homologs allowed us to identify a potential LATLNDIERFL and zinc finger pattern in N-terminal part of pUL56. Recombinant viruses were generated with specific serine or alanine substitutions in these putative patterns. We identified a LATLNDIERFL pattern characteristic of LAGLIDADG homing endonucleases and a metal-binding pattern involving the cysteine and histidine residues C191-X2-C194-X22-C217-X-H219 (CCCH) close to the region conferring letermovir resistance. These patterns are crucial for viral replication, suggesting that they are essential for pUL56 structure and function. Thus, these patterns represent potential targets for the development of new antivirals such as small molecules or peptides and may allow to better understand the letermovir mechanism of action.
Asunto(s)
Citomegalovirus/enzimología , Farmacorresistencia Viral/genética , Endodesoxirribonucleasas/genética , Proteínas Estructurales Virales/genética , Acetatos/farmacología , Secuencia de Aminoácidos , Antivirales/farmacología , Citomegalovirus/genética , Citomegalovirus/fisiología , Empaquetamiento del ADN , Humanos , Dominios Proteicos , Quinazolinas/farmacología , Alineación de Secuencia , Replicación Viral , Dedos de Zinc/genéticaRESUMEN
Merkel cell polyomavirus (MCPyV) is the first polyomavirus clearly associated with a human cancer, i.e. the Merkel cell carcinoma (MCC). Polyomaviruses are small naked DNA viruses that induce a robust polyclonal antibody response against the major capsid protein (VP1). However, the polyomavirus VP1 capsid protein epitopes have not been identified to date. The aim of this study was to identify the neutralizing epitopes of the MCPyV capsid. For this goal, four VP1 mutants were generated by insertional mutagenesis in the BC, DE, EF and HI loops between amino acids 88-89, 150-151, 189-190, and 296-297, respectively. The reactivity of these mutants and wild-type VLPs was then investigated with anti-VP1 monoclonal antibodies and anti-MCPyV positive human sera. The findings together suggest that immunodominant conformational neutralizing epitopes are present at the surface of the MCPyV VLPs and are clustered within BC and EF loops.
Asunto(s)
Proteínas de la Cápside/inmunología , Epítopos/inmunología , Poliomavirus de Células de Merkel/inmunología , Dominios y Motivos de Interacción de Proteínas/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Línea Celular , Reacciones Cruzadas/inmunología , Mapeo Epitopo , Femenino , Humanos , Epítopos Inmunodominantes/inmunología , Poliomavirus de Células de Merkel/genética , Ratones , Modelos Moleculares , Mutación , Conformación ProteicaRESUMEN
PURPOSE: A new human polyomavirus, Merkel cell polyomavirus (MCV), was identified in 2008 in tumor tissue of patients with Merkel cell carcinoma (MCC), a relatively rare human skin cancer. In this study, we investigated patients with MCC and controls for the presence of antibodies against MCV and their association with clinical characteristics. PATIENTS AND METHODS: Antibodies against MCV were investigated by enzyme-linked immunosorbent assay in 68 patients with MCC and 82 controls using VP1 virus-like particles produced in insect cells. RESULTS: Antibodies against MCV were detected in all patients with MCC and in 85% of controls. However, high antibody titers (> 10,000) were rarely observed in controls (7.3%) and they were detected in 64.7% of patients with MCC (P < .001) in contrast to the absence of VP1 expression in tumor samples. In addition, the geometric mean titer of anti-MCV in patients with MCC was around 14 times higher than that observed in MCV-positive controls (P < .001) and was not correlated with tumor viral load. High antibody titers were not found to be associated with any subject or tumor characteristics, but better progression-free survival was observed in patients with high antibody titers (hazard ratio, 4.6; 95% CI, 1.7 to 12.2; P = .002). CONCLUSION: High titers of MCV antibodies in a much higher proportion of patients with MCC than in controls confirmed the association between MCV infection and MCC. The findings also indicated that a better progression-free survival occurred in patients with high MCV antibody titers and suggested that there are at least two distinct etiologic causes of MCC.