ABSTRACT
The purpose of the study was to test the hypothesis that neutrophils can injure cultured skeletal myotubes. Human myotubes were grown and then cultured with human blood neutrophils. Myotube injury was quantitatively and qualitatively determined using a cytotoxicity (51Cr) assay and electron microscopy, respectively. For the 51Cr assay, neutrophils, under non-in vitro-stimulated and N-formylmethionyl-leucyl-phenylalanine (FMLP)-stimulated conditions, were cultured with myotubes at effector-to-target cell (E:T) ratios of 10, 30, and 50 for 6 h. Statistical analyses revealed that myotube injury was proportional to the E:T ratio and was greater in FMLP-stimulated conditions relative to non-in vitro-stimulated conditions. Transmission electron microscopy, using lanthanum as an extracellular tracer, revealed in cocultures a diffuse appearance of lanthanum in the cytoplasm of myotubes and a localized appearance within cytoplasmic vacuoles of myotubes. These observations and their absence in control cultures (myotubes only) suggest that neutrophils caused membrane rupture and increased myotube endocytosis, respectively. Myotube membrane blebs were prevalent in scanning and transmission electron micrographs of cultures consisting of neutrophils and myotubes (E:T ratio of 5) and were absent in control cultures. These data support the hypothesis that neutrophils can injure skeletal myotubes in vitro and may indicate that neutrophils exacerbate muscle injury and/or delay muscle regeneration in vivo.
Subject(s)
Cytotoxicity, Immunologic , Muscle, Skeletal/pathology , Neutrophils/immunology , Adult , Biological Assay , Cells, Cultured , Coculture Techniques , Female , Humans , Lanthanum/metabolism , Muscle, Skeletal/drug effects , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Neutrophils/ultrastructureABSTRACT
Technical parameters of electron microscope staining procedures (type of stain, pH of stain, and time of staining) influence particle integrity for three groups of rotaviruses. Simian rotavirus SA11 (group A), Chinese adult diarrhea rotavirus and porcine rotavirus-like agent (group B), and porcine pararotavirus (group C) were tested. All rotavirus strains were quite stable in uranyl acetate and phosphotungstic acid at pH 4.5 and relatively stable in ammonium molybdate. However, staining with phosphotungstic acid at higher pH values with increased staining time yielded a reduction in the number of particles and particles that were broken or degraded to single-shelled particles or core particles. The different staining procedures were also tested in immunoelectron microscopy experiments. Antibody molecules bound to rotavirus particles were observed clearly only with phosphotungstic acid staining and not with uranyl acetate. We therefore recommend that uranyl acetate and phosphotungstic acid at pH 4.5 be used for negative staining of rotaviruses; phosphotungstic acid at pH 4.5 is optimal for immunoelectron microscopy. These technical points may be critical for rotavirus detection and are important for studies pertaining to the epidemiology and clinical importance of the non-group A rotaviruses.
