Serum deprivation improves seeding and repopulation of acellular matrices with valvular interstitial cells

Cell-extracted valvular tissues (acellular scaffolds, or aScaffolds) offer unique advantages over synthetic polymers for cardiac valve engineering applications in that they retain extracellular matrix molecules to support cellular ingrowth. The extracellular matrix is important in directing many cellular pathways, such as adhesion, proliferation, migration, differentiation, and survival. However, repopulating this type of scaffold often requires high seeding densities or recurrent cell delivery. The optimization of valvular interstitial cell (VIC) seeding onto aScaffolds is reported herein. VICs (the most prevalent cell type in valve leaflets) have maximal growth in 15-20% serum concentrations on tissue-culture polystyrene. Interestingly, after VIC seeding onto aScaffolds, a reduction of serum content, from 15% serum to 5% or less, was found to increase significantly the number of adherent cells, as well as induce transfer of VICs from a tissue-culture polystyrene surface to the aScaffold. aScaffolds seeded and cultured with periods of reduced serum levels were shown to support and enhance VIC viability and attachment, as well as accelerate VIC migration into the aScaffold, leading to a uniformly repopulated valve leaflet construct after 4 weeks of static culture.     

Secondary cytotoxic cell response to lymphocytic choriomeningitis virus: III. In vivo protective activity of effector cells generated in vitro

Secondary cytotoxic T cells were generated in vitro by culturing WE3-LCM virus-specific memory CBA/H spleen cells with WE3-LCM virus-infected syngeneic peritoneal cells at 37° for 5 days, and were found to be highly potent in reducing virus titres in the visceral organs of syngeneic recipients when transferred 24 h before intravenous virus challenge (e.g. 3.1×10⁶ cells transferred gave approximately 3 logs reduction of virus titre). Protection was measured by titrating spleens for virus in a plaque assay, usually 2 days post virus-challenge. Intravenously administered secondary effectors did not, however, elicit a reduction in brain virus titres by 3 days after intracerebral inoculation of virus. Cells mediating protection were sensitive to treatment with anti-θ ascitic fluid plus complement. Protection occurred only when donors of secondary cells, infected stimulator cells and recipients shared H-2K or H-2D, and there was a similar genetic restriction for these effectors to efficiently lyse virus-infected targets in vitro.

Spleen cells from ectromelia virus-memory mice were cultured with ectromelia virus-infected syngeneic peritoneal `stimulator' cells and generated secondary effector cells which protected syngeneic recipients from subsequent challenge with ectromelia but not LCM virus. However, secondary effector cells derived from in vitro stimulation of spleen cells from WE3-LCM virus-memory mice with WE3-LCM virus-infected syngeneic stimulator cells caused a significant reduction in spleen virus titres in syngeneic recipients challenged with either ectromelia or LCM virus. If WE3-LCM virus-infected stimulator cells were fixed and responder spleen cells were either from very old (6–12 months post-priming) WE3-LCM virus-memory mice, or from ARM-LCM virus-memory mice, this unidirectional cross-protection of ectromelia virus-challenged mice was less pronounced. One explanation for the unidirectional cross-protection was that the transferred secondary effectors contained and released infectious LCM virus (or defective virions) which adsorbed to recipients' spleen cells; these latter cells displayed both LCM and ectromelia virus-specific antigenic patterns after ectromelia virus challenge, rendering them susceptible to specific T-cell mediated lysis. Clear specificity of effector cells was demonstrated at the effector: target level in vitro between LCM virus and ectromelia virus. (Experiments to compare the in vivo activity of secondary effectors with primary effector T cells raised in vivo or in vitro were hampered because of the large amounts of infectious virus in the latter two populations.)

Secondary effectors reduced spleen, lung and liver virus titres when transferred 24 h after virus challenge, but were less efficient in protecting recipients than when given before virus.

     

An anti-actin monoclonal antibody inhibits the zona pellucida-induced acrosome reaction and hyperactivated motility of human sperm.

We report an inhibitory effect of an anti-actin monoclonal antibody (mAb) on the human zona pellucida (ZP)-induced acrosome reaction (AR). Motile sperm were incubated with native human ZP for 2 h in medium containing either the anti-actin mAb, an irrelevant control mAb or cytochalasins B or D (40 micromol/l). Sperm bound to the ZP were recovered and the AR was determined by fluorescein-labelled Pisum Sativum agglutinin. Anti-mouse immunoglobulin G (mIgG) Dynabeads, immunofluorescence and immunogold were used to detect the location of the anti-actin mAb in sperm. The anti-actin mAb significantly inhibited the ZP-induced AR (equivalent to cytochalasins), the ionophore A23187-induced AR and hyperactivation of sperm in medium. After incubation with anti-actin mAb, anti-mIgG beads bound to the head of >50% of sperm recovered after binding to the ZP and 10% of sperm remaining in the medium. The proportion of sperm that bound anti-mIgG beads after recovery from binding to the ZP in the presence of the anti-actin mAb was significantly correlated with the ZP-induced AR in the absence of the antibody. Immunofluorescence and immunogold demonstrated entry of the anti-actin mAb into sperm. This study suggests that the sperm plasma membrane becomes permeable to the anti-actin mAb during capacitation and initiation of the AR.     

Copper distribution and reactivity in serum following administration of cuprous oxide to rats and guinea pigs

Total serum copper levels produced by the administration of a single dose of cuprous oxide (500 mg/kg) both s.c. and orally to rats and guinea pigs are reported 1, 5, 7 and 24 h after administration, together with caeruloplasmin oxidase activities and changes in the sulphydryl group concentrations. The highest serum copper levels were obtained one hour after oral administration to the guinea pig. Both animal species exhibited increased serum copper levels after s.c. administration and this increase persisted for a longer time than after oral administration. Caeruloplasmin oxidase activity varied in a complex manner and sulphydryl group activity was significantly depressed in all cases except after s.c. administration to the guinea pig. The latter results must be treated cautiously since they may indicate interactions between copper and the test procedures. Analysis of serum fractions separated by electrophoresis indicated that the absorbed copper is mainly transported on albumin. The dissolution of cuprous oxide by amino acids and hydrochloric acid and the absorption of cuprous oxide and some related copper complexes by albumin were investigated in vitro. The implications of the results are discussed in terms of their relevance to pharmacological and clinical studies.     

Disconnected optic axons persist in the visual pathway during regeneration of the retino-tectal projection in the frog

Summary In this study, we crushed one optic nerve in the frog Litoria (Hyla) moorei and at intervals thereafter anterogradely labelled optic axons with horseradish peroxidase (HRP). For one series, HRP was applied between the eye and the crush site and in a second series between the crush site and the chiasm. A tectal projection of regenerating axons was seen in both series but, in addition, up to 12 weeks post-crush, the second series displayed an additional projection. Its appearance matched that of the disconnected, but persisting, optic axon terminals which are found after enucleation or optic nerve ligation. We conclude that, in the frog, many disconnected optic axons persist throughout the period of optic nerve regeneration and of restoration of an orderly retino-tectal map.Abbreviation HRP horseradish peroxidase     

Mobilization of calcium by the brief application of oxytocin and prostaglandin E2 in single cultured human myometrial cells.

Intracellular calcium ([Ca2+]i) mobilization was studied in single cultured human myometrial cells in response to the agonists oxytocin and prostaglandin E2 (PGE2) using the fluorescent dye Fura-2. Oxytocin and PGE2 applications were associated with an increase in [Ca2+]i, although there was a marked intercell variation in the amplitude of the agonist-induced response. Removal of extracellular calcium ([Ca2+]o) reduced the oxytocin-induced rise and abolished the PGE2-induced rise in [Ca2+]i, thereby demonstrating that oxytocin but not PGE2 can mobilize intracellular stores of calcium. In nominally calcium-free medium, [Ca2+]i was not increased by PGE2 but subsequent application of oxytocin increased [Ca2+]i, thereby demonstrating that, within a single cell, calcium stores were mobilized by oxytocin and not PGE2. The intracellular calcium stores were completely depleted by a single application of oxytocin and not replenished in the absence of [Ca2+]o. Perfusion with calcium-containing medium for 100 s enabled store refilling. Cell depolarization by 140 mM-K+ caused a transient increase followed by a sustained elevation of [Ca2+]i on which were superimposed small fluctuations. Oxytocin caused an influx of calcium in cells depolarized by K+. This was more marked than that obtained with PGE2.     

Organ injuries associated with femoral fractures: implications for severity of injury in motor vehicle collisions

OBJECTIVE: To determine if motor vehicle collisions (MVCs) resulting in femoral fractures were associated with a different injury severity and pattern of injury compared with crashes in which victims did not sustain femoral fractures. METHODS: Retrospective review of seriously injured motor vehicle occupants admitted to a regional trauma unit (Hamilton General Hospital) during a 69-month period (April 1991 to December 1996) for whom detailed crash details were known. RESULTS: Data for 733 motor vehicle occupants with Injury Severity Scores greater than 12 were available; 112 occupants (15.3%) sustained femoral fractures, and 621 occupants (84.7%) did not sustain femoral fractures. Victims with femoral fractures had a significantly higher mean Injury Severity Score (29.4 compared with 25.3 for non-femoral fracture group; p<0.001). The femoral fracture group had a higher incidence of bowel (p<0.012) and hemopneumothorax (p<0.02) injuries as well as an increased incidence of upper and lower extremity (p<0.001) and pelvic (p<0.05) fractures. CONCLUSION: The presence of a femoral fracture is strongly associated with the pattern and severity of injuries sustained by occupants in MVCs. A high index of suspicion is warranted in identifying associated organ injuries in MVC victims with concomitant femoral fractures.