Plasma membranes: phospholipid and sterol content.

The molar ratios of sterol to phospholipid in plasma membranes of five different types of rat cells range from 0.24 to 1.32. The composition of the plasma membrane of a cell has no fixed relation to that of the mitochondria. Thus the structure of cellular membranes shows both tissue and functional specificity.

Recognition of pertussis toxin by antibodies to synthetic peptides.

Eight synthetic peptides, selected from the amino acid sequence of pertussis toxin (PT) subunits S1, S2, S3 and S4, were assessed for their ability to induce protein-recognizing and neutralizing antibodies. Seven of these peptides, prepared as conjugates of either keyhole limpet haemocyanin or tetanus toxoid, induced significant levels of antibody, all of which reacted with SDS-denatured PT on Western blots. Six of the antibodies bound to PT-coated ELISA plates; this binding was inhibited by homologous peptide antigen. However, none of the antibodies, including those directed against the N-terminus of subunit S1, were able to attenuate in vivo or in vitro toxin-dependent activity. Further investigation revealed that only one antibody, specific for the C-terminus of S1 (peptide Slc, 237-255), could recognize the conformation of native PT in solution. The other five antipeptide antibodies which reacted with PT-coated ELISA plates did not recognize PT when captured onto ELISA plates via either a monoclonal antibody or fetuin, unless the conformation of the toxin had been relaxed by reduction with dithiothreitol. Conversely, the native PT-recognizing response of peptide Slc did not bind the conformationally relaxed PT molecule. From this study, it appears likely that a peptide capable of inducing PT-neutralizing antibody must closely resemble the conformation of the cognate sequence in the native protein.

A microplate culture method for assay of guinea pig mitogenic factor.

An improved method for assay of guinea pig mitogenic factor (MF) is described. The technique is based on the use of thymocytes in microplate cultures and yields significant savings in time, labour and materials. The quantitative potential of the assay is examined using antigen- and concanavalin A-indiced MF, and its application to assessing in vitro immune responsiveness of individual guinea pigs is explored.

Studies on the specificity of the vaccine effect elicited by inactivated simian immunodeficiency virus.

Inactivated, partially purified simian immunodeficiency virus (SIVmac) protected macaques from intravenous challenge with homologous and heterologous strains of SIV that had been grown on human cells but no protection against challenge with monkey peripheral blood mononuclear cell-grown SIVmac was afforded. Human immunodeficiency virus type 1 prepared in an analogous way to the SIVmac vaccine on the C8166 human T cell line protected macaques against challenge with human cell-grown SIVmac. These results suggest that protection may be mediated by xenoimmunization with the vaccine cell substrate proteins. All vaccinated macaques had anti-cell antibodies. Major reactivity to MHC class I antigens was found as well as to a 70-kD protein detectable only under nonreducing conditions.

Hormonal and metabolic profiles in subjects with obstructive sleep apnea syndrome and the acute effects of nasal continuous positive airway pressure (CPAP) treatment.

Nocturnal secretion of growth hormone is impaired in patients with obstructive sleep apnea (OSA), but the metabolic consequences have not been reported. We measured blood levels of the hormones insulin, C-peptide, growth hormone, cortisol and glucagon together with the intermediary metabolites of carbohydrate (glucose, pyruvate, lactate, alanine) and lipid metabolism [glycerol, nonesterified fatty acids (NEFA), 3-hydroxybutyrate] in six obese nondiabetic men with OSA on two nights. In the first study, the untreated subjects showed frequent apneas and consequent hypoxemia. The hormone and metabolite concentrations were compared with those obtained on the following night when the subjects were treated effectively with nasal continuous positive airway pressure (CPAP). There were no significant differences in the concentrations of insulin, C-peptide, cortisol or glucagon. We confirmed a marked reduction in growth hormone concentrations in OSA, with a significant increase on the CPAP night. The nocturnal profiles of glucose, pyruvate, lactate, alanine and glycerol showed no differences between the two nights, but concentrations of NEFA and 3-hydroxybutyrate, both products of lipolysis, were significantly greater on the treatment night. Because growth hormone has a lipolytic action, the results suggest that suppression of secretion of growth hormone in untreated OSA results in impaired lipolysis, which is rapidly reversed by nasal CPAP.

Rapid diagnosis of whooping cough using monoclonal antibody.

A counterimmunoelectrophoresis (CIE) method for antigen detection using monoclonal antibody was assessed for its ability to aid in the rapid diagnosis of Bordetella pertussis in 59 patients. A positive diagnosis from a combination of results from tests of serum and urine was obtained in 51 (87%) of cases. For sera, CIE had a sensitivity of 85% and a specificity of 94%; for urine samples the sensitivity was 81% and a specificity of 100%. Antigen detection by CIE is simple to perform and yields results on the same day, thus allowing treatment to begin at an early stage.

Automated colorimetric estimation of glycosylated haemoglobins.

The measurement of glycosylated haemoglobin is now widely used as a guide to glycaemic control in patients with diabetes mellitus. Present methods for measurement of glycosylated haemoglobin are either laborious, expensive or both. We describe a precise and inexpensive method for the automated analysis of glycosylated haemoglobin based on a colorimetric technique. The within-batch coefficient of variation ranged from 2.0 to 4.8% and between-batch was less than 11.0%. At least 200 samples can be analysed per day.

Persistence of antibody after accelerated immunisation with diphtheria/tetanus/pertussis vaccine.

OBJECTIVE: To determine the persistence of antibody to diphtheria, tetanus, and pertussis in children receiving an accelerated schedule of primary immunisation. DESIGN: Controlled study of antibody testing of blood samples from children immunised according to various schedules: three doses of triple vaccine completed at 8-13 calendar months, 6-7 calendar months, before 6 calendar months, or three doses followed by diphtheria/tetanus before age 2. SETTING: Plymouth Health Authority. SUBJECTS: 129 children aged 4 years who had received three doses of diphtheria/tetanus/pertussis vaccine with or without a diphtheria/tetanus booster. MAIN OUTCOME MEASURES: Diphtheria and tetanus antitoxin concentrations and antibody titres to pertussis toxin, filamentous haemagglutinin, and agglutinogens 2 and 3. RESULTS: All children had protective concentrations of antitoxin to diphtheria and tetanus (greater than or equal to 0.01 IU/ml). There was no evidence of a significant difference in diphtheria or tetanus antitoxin concentrations and pertussis antibody titres in children immunised with an accelerated course (third dose of triple vaccine before 6 months) compared with those who received a longer course (third dose at 8-13 months) with no booster (geometric mean antitoxin concentration 0.411 (95% confidence interval 0.273 to 0.618) v 0.426 (0.294 to 0.616) for diphtheria and 0.358 (0.231 to 0.556) v 0.299 (0.197 to 0.453) for tetanus; geometric mean antibody titres 903 (500 to 1631) v 1386 (848 to 2266) for pertussis filamentous haemagglutinin, 179 (130 to 248) v 232 (167 to 322) for pertussis toxin, and 2002 (1276 to 3142) v 3591 (2220 to 5809) for agglutinogens 2 and 3). CONCLUSION: Immunisation with three doses of triple vaccine at monthly intervals completed before 6 months of age probably provides adequate protection against diphtheria, tetanus, and whooping cough which will persist until the age of the preschool booster.

Agglutinogens and fimbriae of Bordetella pertussis.

Agglutinogen 2 (AGG2) of Bordetella pertussis is a fimbrial antigen and therefore a potential adhesin and acellular vaccine component. AGG2 was found to dissociate only under harsh conditions into the subunits of mol. wt. 22500 seen in SDS-PAGE. Results from studies of agglutinogen 3 (AGG3) are presented which confirm previous findings from this Laboratory that AGG3 is also a fimbrial protein but with a subunit mol. wt. of 22000. The amino acid sequence of AGG2, deduced from the nucleotide sequence of the gene encoding it, was used as a basis for synthesis of three peptides. Coupled to Keyhole Limpet Haemocyanin (KLH), the peptides were immunogenic in mice, inducing antibodies which bound well to homologous peptide in ELISA but poorly to intact fimbriae. Monoclonal and polyclonal serotype-specific antibodies failed to react significantly with the peptides or their KLH-conjugates. These results indicate that the synthetic peptides do not represent the serotype 2 epitope. Mice immunized with purified AGG2 or AGG3 were found to be protected against respiratory infection with B. pertussis. Results presented here indicate that this protection is, to a large extent, serotype-specific and that immunization of mice with AGG2 or AGG3 can lead to a change in serotype of the infecting strain. These results are analogous to findings from epidemiological studies of the protection induced in children by whole cell vaccines. They reaffirm the importance of both AGG2 and AGG3 as components of whole cell and acellular vaccines.

Relationship between mitogenic factor and in vitro lymphocyte stimulation during guinea pig immune response to KLH.

Lymphocyte stimulation and production of the lymphokine mitogenic factor (MF) in response to specific in vitro challenge were studied in parallel using lymphocytes from animals sensitized with different doses of keyhole limpet haemocyanin at various times after sensitization. Overall, agreement between the two assays of cellular response was good. However, examination of the antigen dose-response characteristics in each experimental situation revealed dissociation in some cultures. Blood leucocyte responses developed later than those of lymph node cells. The latter gave good MF but were poorly stimulated at day 60, when both responses were maximal for blood leucocytes, indicating a possible redistribution of responsive cells from the nodes to the blood. Treatment of guinea pigs with cyclophosphamide affected lymphocyte stimulation to a greater extent than MF production. Only a part of the antigen-induced stimulation of lymph node cells could be attributed to MF elicited in culture.

Mitogenic factor as an in vitro correlate of delayed hypersensitivity in the guinea pig.

The relationship of mitogenic factor (MF) to delayed hypersensitivity was examined in the guinea pig. MF was readily produced in vitro by blood leucocytes from animals sensitized with Keyhole limpet haemocyanin in Freund's complete adjuvant. Such animals exhibited delayed skin reactions and macrophage migration inhibition. Treatment with cyclophosphamide markedly reduced serum antibody but caused no diminution of MF or delayed skin reactions. A migration inhibition response was observed 10, but not 20, days after sensitization by means of incomplete adjuvant.

Effects of variation in the level of haptenation of heterologous protein immunogen on cellular and humoral responses of the guinea pig.

Groups of guinea pigs were sensitized with bovine gamma-globulin (BGG) substituted with 0, 4, 12, or 40 dinitrophenyl (DNP) groups per molecule. Skin tests, lymphocyte stimulation, two lymphokine assays and antibody determinations were carried out after 21 days using all four antigens. In the extreme situations when BGG-sensitized animals were challenged with DNP40-BGG, or when DNP40-BGG-sensitized animals were challenged with BGG, the delayed skin reactions were transient and weakly indurated. BGG-sensitized guinea pigs injected with anti-DNP40-GBB serum showed no change in delayed skin response to DNP40-BGG, whereas anti-BGG serum enhanced the response of DNP40-BGG-sensitized anamals to BGG. All immunized groups showed lymphocyte stimulation and lymphokine production by all antigens, but migration inhibition and lymphocyte stimulation followed less closely the pattern of delayed skin responses than did mitogenic factor output. The serum antibody response to DNP40-BGG as immunogen indicated that all carrier determinants were masked. At lower levels of substitution, anti-hapten titres were reduced and appreciable levels of anti-carrier antibody obtained.

The relationship between the serogroup antigen and lipopolysaccharide of Legionella pneumophila.

Serogroup-specific antigen was extracted from a number of Legionella pneumophila strains and compared with phenol-water extracted lipopolysaccharide on the basis of gel filtration, chemical analysis, SDS-PAGE and reaction with serogroup-specific antibody in immunoblots. Serogroup specificity is apparently borne by the O side-chains of the lipopolysaccharide, which, although smooth in type, partitions in the phenol phase. For four serogroup 1 strains tested, there was no qualitative correlation between O side-chain length and pulmonary virulence for guinea-pigs.

Heterogeneity of the filamentous haemagglutinin of Bordetella pertussis studied with monoclonal antibodies.

The filamentous haemagglutinin of Bordetella pertussis has been purified from static, liquid culture supernatants and from extracts of cells grown on a solid medium. SDS-PAGE of the purified protein has shown multiple polypeptides with molecular weights ranging from 220 000 to about 58 000. By transferring the SDS-dissociated polypeptides to nitrocellulose paper and reacting with several monoclonal antibodies, it has been shown that many of the polypeptides are probably fragments of the polypeptide of highest molecular weight.