Herpes simplex virus type 1 morphogenesis and virus-cell interactions: significance of cytoskeleton and methodological aspects.
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Cell Communication↗
Biomedical subjects
Helle Lone Jensen
Publications and source records attributed to Helle Lone Jensen.
Herpes simplex virus-cell interactions studied by low-fading contrasted immunofluorescence.
The low-fading immunofluorescence with propidium iodide contrast described here is recommended for light and confocal viral antigen identification and other cell biology studies because: (1) it is a simple, rapid, sensitive, and reproducible technique; (2) phase-contrast microscopy is unnecessary; (3) contrast is optimal without blurring the fluorescent labeling; (4) autofluorescence is minimal, even in fixed cells; (5) background staining is minimal; (6) fading is invisible for at least 5-min exposures, even in preparations with weak antigen presentation; (7) fluorescence is stable after storage in the dark at -20 degrees C; (8) fluorochromes are small-sized markers without steric hindrance; and (9) there is no need for silver enhancement or substrate solutions, which increase the risk of diffusion and other artifacts.
Clinical Laboratory Techniques↗
Herpes simplex virus-cell interactions studied by immunogold cryosection electron microscopy.
A technique is presented for high-resolution postembedding immunolocalization of one or two (or several) antigens in the same ultrathin cryosection using primary monoclonal antibodies from the same species. The optimized three-layer indirect immunogold-labeled cryosection electron microscopy described is recommended for studies of virus-cell interactions, because: (1) it is a simple and reproducible method; (2) colloidal gold markers are electron-dense, stable, and easy to recognize; (3) the membraneous ultrastructure and immunolabeling are well preserved; (4) immunolabeling is less in the two-layer method; (5) silver-enhanced gold particles vary in size and shape; (6) it is possible to demonstrate herpes simplex virus type 1 glycoproteins gC-1 and gD-1 in the nuclear membranes and gC-1- and gD-1-labeled viral particles in the perinuclear space and to observe virions in the endoplasmic reticulum and Golgi area. The use of buffered 3% paraformaldehyde plus 2% glutaraldehyde for 2 h at room temperature effectively destroys free anti-IgG binding sites on the secondary antibodies in double-labeling immunogold cryosection electron microscopy and is recommended because: (1) inactivation is obtained through buffered primary fixative; (2) the method is simple and reproducible; (3) cross-labeling is effectively avoided; (4) silver-intensification, high temperature, and methyl cellulose cover of ultrathin cryosections are avoided between the staining sequences; and (5) ultrastructure and antigenicity are well preserved.
Epithelial Cells↗
Acute fatal pulmonary vein occlusion after catheter ablation of atrial fibrillation.
BACKGROUND: In treatment of atrial fibrillation (AF) catheter radiofrequency isolation of the pulmonary veins (PVs) has proved to be highly successful. There have been several case reports regarding PV stenosis, however none of these have reported a fatal outcome. METHODS AND RESULTS: A 31-year-old man was referred to us for treatment of complications related to catheter ablation. According to the documentation from the hospital, the patient underwent segmental ostial PV isolation for treatment of AF. A few hours after the procedure, the patient developed dyspnoea, hemoptysis, and a high fever. The patient was first diagnosed as having pneumonia but five days later transesophageal echocardiography and pulmonal angiography revealed total occlusion of the left superior and inferior PVs. When we received the patient he underwent open-heart surgery, which showed thrombi in the orifices of the left sided PVs protruding into the left atrium. In each of the left sided PVs severe stenosis was seen in the bifurcation area. Thrombus material was removed followed by placement of two stents in each of the left sided pulmonary veins at the first bifurcations. However, the patient died 14 days after the ablation procedure. Selective autopsy of the left lung revealed diffuse alveolar damage, disseminated intravascular coagulation, multiple thrombi formation, and haemorrhagic infarctions. CONCLUSIONS: PV stenosis may occur very early after the ablation procedure. Delayed diagnosis can be fatal. The early stenosis may result in thrombus formation in the left atrium and PVs and in this case surgery should be considered.
Adult↗
The morphogenesis of herpes simplex virus type 1 in infected parental mouse L fibroblasts and mutant gro29 cells.
Mutants of cell lines and viruses are important biological tools. The pathway of herpesvirus particle maturation and egress are contentious issues. The mutant gro29 line of mouse L cells is defective for egress of herpes simplex virus type 1 (HSV-1) virions, and a candidate for studies of virus-cell interactions. The properties of uninfected and HSV-1-infected L fibroblasts and gro29 cells investigated by protein assay, immunoblot, titration assay, immunofluorescence light microscopy and immunogold cryosection electron microscopy are reported. The ultrastructure of both HSV-1-infected L and gro29 cells confirmed primary envelopment of virions at the nuclear membranes followed by maturing multiple de-envelopments and re-envelopments in the endoplasmic reticulum and in the Golgi complex. The gro29 cells presented changed cytoskeleton, abolished egress of virions, and were defective in the trafficking of glycoproteins, giving rise to accumulation of viral particles and glycoproteins in the endoplasmic reticulum and the Golgi complex. The results suggest that gro29 cells harbour a causal underlying defect of the cytoskeleton in addition to the HSV-1-induced cytoskeletal changes.
Actins↗