Interaction of Pseudomonas cytochrome cd1 with the cytoplasmic membrane.

Labelling with ferritin-conjugated antibody shows that Pseudomonas cytochrome cd1 is associated with the inner surface of the cytoplasmic membrane. Cytochrome cd1 is however, enriched to the soluble fraction obtained after destruction of Pseudomonas spheroplasts. Comparison of the respiratory nitrite reductase activities, due to this cytochrome, between different cellular fractions and the purified enzyme shows that while the kinetic pattern and the temperature dependence of the activity remain almost the same the molecular activity is enhanced when the enzyme is released from cells. A new assay of respiratory nitrite reductase was developed in this study. The method is based on determination of the stoichiometrical proton consumption accompanying nitrite reduction.

The quatenary structure of Pseudomonas cytochrome oxidase studied by electron microscopy.

Pseudomonas cytochrome oxidase (EC 1.9.3.2) was studied by negative staining in the electron microscope. The best resolution was obtained with uranyl oxalate (pH 6.0) as negative stain. Electron micrographs confirm the idea of the dimeric structure of the enzyme. A rough model of cytochrome oxidase was constructed based on different projections of the molecule seen in the electron micrographs. In this model the subunits are identical and sterically equivalent.

The subunit structure of Pseudomonas cytochrome oxidase.

Pseudomonas cytochrome oxidase (EC 1.9.3.2) is composed of two subunits. Each subunit has a molecular weight of approx. 63000 and, according to the iron determination, contains two hemes. Cytochrome oxidase was subjected to various dissociation procedures to determine the stability of the dimeric structure. Progressive succinylation of 14 to 68% of the lysine residues of the enzyme increases the amount of the protein appearing in the subunit form (S20,W approximately 4 S) from 18 to 92%. At a high degree of succinylation a component with a sedimentation coefficient of approx. 2 S appears. The subunits with sedimentation coefficients of approx. 4 S and 2 S are also formed when the pH is below 4 or above 11. The same molecular weight (63000) was found for these two components in sodium dodecylsulphate electrophoresis. No dissociation of cytochrome oxidase was observed in salt solutions like 3 M NaC1 and 1 M Na2SO4, or in 6 M urea. The slight decrease in the sedimentation coefficients in NaC1 solutions is partly explained by preferential hydratation of the protein.

Loss of protective immunity to polio, diphtheria and Haemophilus influenzae type b after allogeneic bone marrow transplantation.

Immunity to poliovirus, diphtheria and Haemophilus influenzae type b (Hib) was studied in 16 adult recipients of a bone marrow transplant from an HLA-identical sibling donor in order to evaluate the need for revaccinations. T-cell depletion was not done in any case. The donors and patients were studied before bone marrow transplantation (BMT) and the patients 1, 3, 6, and 12 months later. Prior to the BMT 10 of 11 patients were immune (titre > or = 4) to all vaccine poliovirus types by a standard microneutralization assay. At 12 months after BMT only two of seven patients were immune to all vaccine types, and none had immunity against an antigenically altered poliovirus type 3 strain Finland. The geometric means of antibody titres against poliovirus types 1, 2, and 3 strain Saukett and strain Finland declined gradually after 1 month postgrafting, being 4.4, 5.4, 3.3, and 1.3 respectively at 12 months after BMT. At 1 year 6 of 11 patients had immunity against diphtheria by a toxin neutralization method, but the antitoxin geometric mean level had decreased to a barely protective level, 0.01 IU/ml. The geometric mean Hib antibody concentration decreased during the first 6 months after BMT and thereafter increased slightly. A significant proportion of BMT recipients lose their protection against polio, diphtheria and Hib, and revaccinations are necessary.

Diagnosis of pertussis.

During a local outbreak of pertussis mainly involving school-children and adults, 71 patients with suspected whooping cough and 25 of their household contacts were studied in the course of 1 day so as to compare enzyme-linked immunosorbent assay (ELISA) with bacterial isolation in the diagnosis of pertussis. Serum samples and two nasopharyngeal swabs were collected from all persons. Five different culture media were used. Twenty-eight cases were diagnosed within 2 days by measuring IgM- and IgA-class antibodies to Bordetella pertussis in the first serum samples of patients. Fifty cases were identified when paired serum samples were used. Cultures were positive in only nine patients. The results show that ELISA is a valuable aid in the diagnosis of pertussis in patients with negative cultures.

Observations on a formaldehyde low-temperature steam sterilizer.

In Finnish hospitals, heat labile equipment is mostly sterilized by ethylene oxide (EO) gas. Formaldehyde sterilizers are rarely used. We have tested a new commercial autoclave constructed exclusively for formaldehyde and low-temperature steam (F-LTS) sterilization, offering a potentially safer and cheaper method of sterilization. Both the sterilizing efficacy of the apparatus and the levels of formaldehyde in the processed materials were studied.

High-performance liquid chromatography in the quality control of immunoglobulin preparations during production and storage.

High-performance liquid chromatography (HPLC) has been used in the routine quality control of immunoglobulin preparations to measure the contents of aggregates, dimers, monomers and degradation products. The correlation coefficients between TSK 3000 SW, Ultrogel AcA 34 and Sephacryl S-300 chromatography were calculated and a reasonable correlation was found. The decrease in the potency of anti-tetanus immunoglobulin at room temperature was accompanied by an increase in degradation products. The speed and high sensitivity of HPLC make it suitable for the detection of aggregates in intravenous immunoglobulin preparations.

Class-specific antibody response to lymphocytosis promoting factor (LPF) and fimbriae (F) in pertussis.

IgM, IgA and IgG antibodies to lymphocytosis promoting factor and fimbriae were measured by enzyme-linked immunoassay in seropositive and/or culture-positive patients without symptoms of pertussis, culture-negative patients with typical whooping cough symptoms, and culture-positive patients with typical symptoms. The culture-negative patients with typical symptoms had higher antibody levels in the first specimens and stronger responses to these antigens than the other patients. Antibody levels in the first specimens and antibody responses did not correlate with patients' age and duration of symptoms before taking of the first specimens. Thus, no correlation occurred between the symptoms of pertussis and antibody levels or antibody responses to fimbriae or lymphocytosis promoting factor. Bacteria were less likely to be isolated from those with strong antibody responses and this stresses the need of using both isolation and serology in the diagnosis of pertussis.

Immunity to diphtheria in Helsinki in 1975.

To determine the status of the population's immunity to diphtheria, the Schick test was performed on 489 and the diphtheria antitoxin level determined in 404 hospital patients, all residents of Helsinki. A low ("unprotective", less than 0.01 IU/ml) antitoxin level was found in not more than 5% among persons under 20. The highest percentage, 48%, was found in the 50--59 years age group. A low antitoxin level was more common among women than men in those over 40 years of age. Taking into consideration the age structure of the population of Helsinki in 1975, 23% of the population had an antitoxin level less than 0.01 IU/ml. The percentage of Schick-positives among different age groups was almost the same as the percentage of low antitoxin levels. Of 100 serum samples from military recruits coming from different regions of the country, 15% had an antitoxin level below 0.01 IU/ml.

Serologic diagnosis of pertussis: comparison of enzyme-linked immunosorbent assay and bacterial agglutination.

An enzyme-linked immunosorbent assay (ELISA) and bacterial agglutination (BA) method for determining the presence of antibodies to Bordetella pertussis were compared on serum samples from 21 patients with whooping cough and their 76 family members. The overall diagnostic agreement between the two methods was 77%. The data for BA-detected antibodies correlated best with IgG and IgA antibodies to B. pertussis. All of the culture-positive patients showed serologic positivity in both assays during the follow-up. Pertussis was diagnosed by ELISA in most cases from the first serum sample. Both methods proved to be good diagnostic aids in culture-negative patients, although the value of BA is more retrospective because of the need for paired sera. The kinetics of IgM, IgA, IgG, and agglutinating antibodies to B. pertussis is presented.

A rapid and simple method for evaluation of serum opsonic capacity against Bordetella pertussis by chemiluminescence.

Serum opsonic capacity against Bordetella pertussis was studied by using quantitative chemiluminescence (CL) which is known to have several advantages over conventional methods in the evaluation of opsonization and phagocytosis. For opsonization, sera from whooping cough patients or from controls were incubated with killed B. pertussis cells at 37 degrees C for 30 min. Polymorphonuclear leukocytes, mononuclear cells or unfractionated leukocytes, all from healthy blood donors, were added to the opsonization mixture and CL emission was measured. Bacteria opsonized in diluted sera of whooping cough patients caused the activation of leukocytes manifested as a CL emission and no CL emission was observed when unopsonized B. pertussis was incubated with leukocytes. The opsonins could be detected by using any type of leukocyte preparation. Sera from adults vaccinated with DTP in the childhood gave rise to a large CL emission. Sera from whooping cough patients produced CL responses in more dilute solutions than those from vaccinated controls and sera from unvaccinated infants did not contain opsonins against B. pertussis. In unvaccinated infants suffering from whooping cough no opsonins were detectable in sera collected one week after the onset of the disease. However, the development of the opsonic capability against B. pertussis was observed in follow-up sera of all patients. The CL assay is simple, rapid, and reproducible offering new possibilities to evaluate humoral immune mechanisms and phagocytosis in whooping cough.

Serum antibody response to B. pertussis Tn5 mutants, purified PT and FHA in two different mouse strains and passive protection in the murine intranasal infection model.

The protective capacities of antibodies to pertussis toxin (PT) were compared with antibodies to several other pertussis antigens in an experimental murine model of intranasal infection with Bordetella pertussis. Protection from lethal challenge was achieved by passive immunization with mouse antisera to whole cells of the virulent B. pertussis BP338(Vir+) and its Tn5-generated mutants, BP353(Fha-), BP348(Adc-Hly-) and to a lesser extent of BP347(Vir-). The immune sera were produced in two different mouse strains, a good PT antibody responder (NIH) and a poor responder (F1 of CBA x C57BL/6). The antisera produced in the F1 mice contained no detectable neutralizing antibodies to PT as measured by the CHO cell assay. In spite of this the anti-BP353(Fha-) and BP348(Adc-Hly-) sera of the F1 mice seemed as protective as those of the NIH mice. A strong dependence between PT neutralizing antibody and protection was seen only when comparing sera of NIH and F1 mice immunized with purified active PT. The protective capacity of sera of both mouse strains immunized with purified filamentous hemagglutinin (FHA) correlated with their anti-FHA titers measured by enzyme immunoassay. The data thus confirm the protective capacity of anti-PT and anti-FHA, but also show that antibodies of other specificities can confer protection.

Meningococcus group A vaccine in children three months to five years of age. Adverse reactions and immunogenicity related to endotoxin content and molecular weight of the polysaccharide.

Vaccination of 21,007 children between the ages of three months and five years was completed with five different lots of the meningococcal group A capsular polysaccharide vaccine. A correlation was found between the frequency and severity of adverse reactions and the endotoxin content of the vaccine lots. All vaccine lots elicited a serum antibody response. The endotoxin content of the vaccines did not correlate with the serum antibody response.

Serologic diagnosis of pertussis: evaluation of pertussis toxin and other antigens in enzyme-linked immunosorbent assay.

IgM, IgA, and IgG antibodies to Bordetella pertussis were measured in paired sera from 34 patients who were culture-positive for pertussis by enzyme-linked immunosorbent assay (ELISA) with disrupted B. pertussis bacteria, purified pertussis toxin, or outer membrane proteins (OMP) as antigens. Paired sera from 50 patients with other respiratory infections were used as controls. The sensitivities of the assays from paired sera were 61%, 90%, and 90% and specificities were 98%, 92%, and 72%, respectively. Of the patients culture-positive for pertussis, 68% had positive levels of antibody to pertussis toxin antigen in their first serum samples, obtained at the same time as samples for culture. Infants had antibody responses to pertussis toxin antigen, in contrast to weak antibody responses measured by B. pertussis antigen. The results from this study indicate that ELISA, especially measuring pertussis toxin IgA, is a valuable additional tool for diagnosing pertussis and can be used as a complementary test with cultures.

Adverse reactions and endotoxin content of polysaccharide vaccines.

Adverse reactions occurring when 1.5 million children and young adults were vaccinated with different lots of group A meningococcal and one lot of Haemophilus influenzae type b capsular polysaccharide vaccines were analyzed on the basis of records kept by local health personnel and 19,000 special questionnaires. The incidence of anaphylactic reactions was 0.8 per 100,000 injections. A pyrogen-like reaction--high fever appearing soon after injection and disappearing within six hours--was seen in 1.8% of children 3 months to 5 years old who received the meningococcal vaccine in the first vaccination project. This reaction was most common in children below three years. Its frequency varied with different vaccine lots and was found to be correlated with the endotoxin content of the vaccines estimated by either the limulus assay or by pyrogen test in rabbits. On the basis of these results we propose that capsular polysaccharide vaccines should pass the pyrogen test with a dose of one mug polysaccharide per kg of rabbit weight. With such vaccines, the incidence of high fever was reduced to 0.5% in children of the same age group.

Outbreak of paralytic poliomyelitis in Finland: widespread circulation of antigenically altered poliovirus type 3 in a vaccinated population.

An outbreak of 9 cases of paralytic poliomyelitis and 1 non-paralytic case occurred in Finland between August, 1984, and January, 1985, after two decades of freedom from the disease attributable to a successful immunisation programme. During the outbreak poliovirus type 3 was isolated from the patients, from about 15% of healthy persons tested, and from sewage water. At least 100 000 persons were estimated to have been infected. With 1.5 million extra doses of inactivated poliovirus vaccine to children under 18 years of age and an oral poliovirus vaccine campaign covering about 95% of the entire population in February-March, 1985, the outbreak was halted in February, 1985. Impaired herd immunity to the epidemic strain of poliovirus type 3, which differed from the type 3 vaccine strains in both immunological and molecular properties, was important in the emergence of this outbreak. The inactivated poliovaccine that had been used in the vaccination programme was relatively weakly immunogenic, especially as regards the type 3 component. Whether continuous antigenic variation of poliovirus type 3 has wider epidemiological implications is not known.