The use of 7-amino actinomycin D in identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques

The detection and quantitation of apoptotic cells is becoming increasingly important in the investigation of the role of apoptosis in cellular proliferation and differentiation. The pathogenesis of hematologic disorders such as aplastic anemia and the development of neoplasia are believed to involve dysregulation of apoptosis. To quantitate accurately the proportion of apoptosis cells within different cell types of a heterogeneous cell population such as blood or bone marrow, a method is required that combines the analysis of large numbers of cells with concurrent immunophenotyping of cell surface antigens. In this study, we have evaluated such a method using the fluorescent DNA binding agent, 7-amino actinomycin D (7AAD), to stain three diverse human cell lines, induced to undergo apoptosis by three different stimuli. Flow cytometric analysis defines three populations on the basis of 7AAD fluorescence and forward light scatter. We have shown by cell sorting and subsequent morphological assessment and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling that the populations defined by 7AAD represent live, apoptotic, and late-apoptotic/dead cells. This method is quick, simple, reproducible, and cheap and will be a valuable tool in the investigation of the role of apoptosis in normal physiology and in disease states.     

Alphavirus replicon particles expressing the two major envelope proteins of equine arteritis virus induce high level protection against challenge with virulent virus in vaccinated horses

Replicon particles derived from a vaccine strain of Venezuelan equine encephalitis (VEE) virus were used as vectors for expression in vivo of the major envelope proteins (G(L) and M) of equine arteritis virus (EAV), both individually and in heterodimer form (G(L)/M). The immunogenicity of the different replicons was evaluated in horses, as was their ability to protectively immunize horses against intranasal and intrauterine challenge with a virulent strain of EAV (EAV KY84). Horses immunized with replicons that express both the G(L) and M proteins in heterodimer form developed neutralizing antibodies to EAV, shed little or no virus, and developed only mild or inapparent signs of equine viral arteritis (EVA) after challenge with EAV KY84. In contrast, unvaccinated horses and those immunized with replicons expressing individual EAV envelope proteins (M or G(L)) shed virus for 6-10 days in their nasal secretions and developed severe signs of EVA after challenge. These data confirm that replicons that co-express the G(L) and M envelope proteins effectively, induce EAV neutralizing antibodies and protective immunity in horses.     

Evaluation of attempted prevention of unexpected infant death in very high-risk infants by planned health care

Three hundred and ninety-six babies born in Sheffield between 1982 and 1990 identified as being at "very high risk" of unexpected infant death by means of a scoring system, received an intensive programme of health care including a case discussion between a paediatrician, the GP and the health visitor held in the family doctor's surgery, weekly visits from the health visitor and informal hospital admission. Significantly fewer sudden unexpected infant deaths occurred in this group than were expected by logistic regression anlysis or occurred in the best available control group with comparable scores ( p = 0.024). Problems in evaluation include identification of an adequate control population, ethical difficulties in introducing a controlled study when the programme is already perceived as effective, and the calculation of "expected death rates". The results of this study indicate that very energetic programmes of intervention may prevent some deaths in vulnerable infants.     

Outcome of congenital lung abnormalities detected antenatally

To determine the outcome of congenital lung abnormalities, data were collected retrospectively between January 1991 and December 1996 on any foetus found to have a lung lesion on antenatal ultrasound. A total of 23 foetuses had lung lesions on antenatal ultrasound. In two foetuses the antenatal ultrasound showed bilateral enlarged "bright" echogenic lungs with evidence of hydrops. Both pregnancies were terminated and tracheal atresia was confirmed. In 15 foetuses the antenatal ultrasound appearance was of a unilateral "bright" echogenic lung. There was one case of bronchial atresia and two cases of congenital lobar emphysema, which all had surgery. In nine cases there was a reduction in the size of the lesion on serial antenatal ultrasounds and no lesion was detected after birth. In three cases a small lesion was present after birth on chest radiography. In six foetuses the antenatal ultrasound appearance was of unilateral cystic or mixed cystic and echogenic lung lesions. Two pregnancies were terminated; both had congenital cystic adenomatoid malformation. Four pregnancies were continued and three infants had surgery soon after birth and were confirmed to have had congenital cystic adenomatoid malformation. One infant has been managed conservatively. In conclusion, a definitive diagnosis cannot usually be made antenatally. A large lesion on initial scan does not necessarily predict a poor outcome. The natural history of small asymptomatic postnatal lesions is unknown and a long-term prospective study is needed to determine the outcome of these lesions.     

Airway intervention in adult supraglottitis

Background The timing of aggressive airway intervention in adult epiglottitis is controversial. Aims To correlate Friedman’s staging of epiglottitis on admission with the airway interventions undertaken. Methods A retrospective study of 23 adult patients, mean age 51 years (range 29–81 years), who had been admitted with acute supraglottitis between March 1988 and December 2000 was undertaken. Results Three patients (13%) had airway interventions; two with tracheostomy and one with tracheal intubation. All were Friedman stage III and had rapid symptom progression during the 24 hours prior to admission. Three other stage III patients with symptom progression longer than 24 hours and all the remaining patients (stage II or less) were managed with observation and intravenous therapy. Conclusions Friedman originally advocated airway intervention in any patient stage II or worse, but this intubation threshold should probably be lowered to those patients with rapid-onset stage III (moderate respiratory distress, stridor, respiratory rate >30 per minute, pCO₂ >45mmHg) disease.