Effect of 2-mercaptoethanol and mycostatin on guinea pig lymphocyte transformation: interpretation of results depends on the method of calculation.

The stimulating effects of 2-mercaptoethanol (2-ME) reported in murine cell cultures were confirmed for guinea pig lymphocyte transformation (LT). 2-ME was mitogenic for peripheral blood lymphocytes (PBL) and lymph node lymphocytes (LNL) and enhanced PPD-induced LT. The effect on LNL was dependent on the magnitude of the response of non-2-ME treated cultures. The 2-ME effect on PPD-stimulated LNL was reversed when stimulation index (SI) replaced the delta cpm estimate. Mycostatin (MYC) inhibited the blastogenic response of PBL control cultures, whereas its effect on LNL control cultures varied. The interpretation of the effect of MYC on PPD-stimulated cultures was dependent on the use of the delta cpm or SI estimates. The interpretation of LT experiments is therefore highly dependent on whether delta cpm or SI is used for expression of results. Analysis of effects on control and stimulated cultures should precede the addition of 2-ME or MYC to cultures for LT.

Statistical evaluation of the lymphocyte proliferation assay with non-stimulated cultures.

A micromethod for the lymphoproliferative assay has been evaluated statistically with non-stimulated human lymphocyte combinations. Different variance components were estimated by means of a hierarchical analysis of variance. Instead of using an arbitrary value for a significant difference between stimulated and non-stimulated cultures, the value can be calculated on the basis of the components of variance in the experimental design. The method disclosed systematic differences between the values of control cultures depending on the lymphocyte combinations and groups of persons examined, which underlines that every experiment should always contain non-stimulated cultures. Further, cell-cell cooperation and differences in spontaneous DNA synthesis within individual lymphocyte populations could be demonstrated.

Estimation of guinea pig antigen-specific and non-specific suppressor cell activity.

A functional assay for the quantitative estimation of suppressor cell (SC) activity in guinea pigs has been developed. Cultures of antigen-stimulated peripheral blood lymphocytes (PBL) from sensitized guinea pigs develop SC activity. The suppression of proliferation can be demonstrated in antigen-stimulated autologous co-cultures of precultured and freshly isolated PBL. The extent of suppression is dependent on the preculture antigen concentration but not the preculture period and it consists, as demonstrated with PBL from doubly sensitized guinea pigs, of an antigen-specific and a non-specific component. The observed SC activities were not due to an alteration of the kinetics of the co-cultures. The estimates of suppression are highly dependent on corrections for the values of the control cultures. The present method may prove useful in immunological studies of mycobacterial infections in guinea pigs.

Production, assay and partial characterization of guinea pig interleukin 2.

Optimum conditions for the production and assay of guinea pig interleukin-2 (IL-2) have been established. The mitogenic activities of serial dilutions of guinea pig IL-2 preparations were compared in cultures of guinea pig peripheral blood lymphocytes prestimulated for 7 days with phytohemagglutinin (PHA) used at 1 microgram/ml. Parallel log dose-log response curves were used for quantitative comparisons. Optimum IL-2 yields were obtained from cultures of lymph node lymphocytes stimulated for 20 h with Concanavalin A (ConA) at 5 micrograms/ml. Guinea pig T cell lines reactive to mycobacterial antigens were propagated for several months using our IL-2 preparations. The molecular weight of guinea pig IL-2 was estimated to be 30,000 using S-200 gel filtration. The species specificities of guinea pig, human, mouse and rat IL-2s were examined. It was shown that guinea pig T lymphocyte blasts were stimulated only weakly with human IL-2 and not at all with mouse and rat IL-2.

Antigen specific in vitro activity of lymphocyte subpopulations from Mantoux positive and negative subjects assessed by lymphocyte proliferation assay.

A micromethod for the lymphocyte proliferative assay and multifactorial analysis of stimulation indices classified according to subjects, lymphocyte combinations and antigens was used to elucidate deficient Mantoux (Mx) reactions in BCG-vaccinated subjects. It was possible to group each of the factors so that interactions between the two latter factors were not present within but only between groups. Comparisons between the subject groups showed a clear association between results of this in vitro test with PPD antigen and the Mx test carried out with 1 TU. The in vitro test had the advantage of being more sensitive and of not introducing the immune reactions occasionally seen with the Mx test. Thymus-processed lymphocytes were the cells responsible with functions both as effector and regulator cells, B lymphocytes had only regulatory properties.

Relation between enzyme-linked immunosorbent assay and radioimmunoassay for detection of antibodies to the capsular polysaccharide of Haemophilus influenzae type b.

The measurement of antibodies to the capsular polysaccharide (PRP) of Haemophilus influenzae type b (Hib) is important because vaccines inducing such antibodies are now available. We developed and evaluated an enzyme-linked immunosorbent assay (ELISA) for detection of these antibodies based on direct coating of the plates with tyraminated PRP. The assay fulfilled the requirements for parallel line assays; it was sensitive, specific, and reproducible with a coefficient of variation between days of 19%. Results from the ELISA were compared with results from radioimmunoassay and a correlation coefficient of 0.93 was found. Results obtained by the two methods were proportional and the relation was independent of the antibody level. The relation between them was also unaffected by the contribution of different antibody isotypes, indicating that these were measured to the same extent by both methods. ELISA employing direct coating of the plates with tyraminated PRP represents a useful alternative for detection of antibodies when studying immunogenicity of Hib vaccines.

Resistance to dicloxacillin, methicillin and oxacillin in methicillin-susceptible and methicillin-resistant Staphylococcus aureus detected by dilution and diffusion methods.

A total of 54 Staphylococcus aureus strains of varying methicillin resistance were investigated for their resistance to methicillin, oxacillin and dicloxacillin by different diffusion tests. Inhibition zones were measured around locally prepared paperdiscs with 10 micrograms methicillin, 5 and 10 micrograms oxacillin, 5 and 10 micrograms dicloxacillin, PDM paperdiscs with 10 micrograms methicillin or oxacillin and Neo-sensitabs tablets with methicillin or oxacillin. All diffusion tests were performed both with Mueller-Hinton agar and Danish Blood agar as well as at 37 degrees C and 30 degrees C and read after overnight incubation. Differences in zone diameter under different conditions were found to be independent of the susceptibility level of the strains. Seventeen of the strains were detected as methicillin-resistant (MRSA) by two methods including high inoculum and prolonged incubation at 30 degrees C. The minimum inhibitory concentration (MIC) of the 54 strains was determined by a plate dilution method at 30 degrees C and 37 degrees C. A 10 micrograms locally prepared methicillin disc detected all MRSA strains with no false reactions either at 37 degrees C or 30 degrees C on Mueller-Hinton agar. Investigations with oxacillin discs had to be performed at 30 degrees C or with a 5 microgram disc in order to detect correctly. PDM paperdiscs gave reactions identical to the corresponding locally prepared discs. Methicillin Neo-sensitabs detected all MRSA strains but also included a few susceptible strains among the resistant ones. Addition of blood increased the number of not-detected MRSA strains. All 17 MRSA strains were susceptible to dicloxacillin by the dilution method, and the disc diffusion test showed similar results. Dicloxacillin discs therefore did not detect the presence of MRSA strains. The implications of replacement of the methicillin/oxacillin disc by a dicloxacillin disc are discussed.

A subdivision of strains of Staphylococcus aureus in the 94,96 complex by means of experimental phages.

In order to facilitate epidemiological investigations a subdivision of Staphylococcus aureus strains belonging to the 94,96 complex by means of two experimental phages, 16 and 47A, was performed. These phages were selected from the nine experimental phages initially examined because they gave the greatest discrimination. On the basis of reactions with these two phages, 2199 isolates which reacted with phages 94 and 96, and 773 isolates which reacted with phage 96 alone, were each subdivided into two major and two minor groups. Strains with different phage patterns were in a few cases (2/64) isolated from the same deep body site in a patient, and lysogenisation experiments suggested that differences in phage patterns were determined by the presence of prophages. Strains with the phage patterns 94/96 and 96 were found to be unevenly distributed throughout Denmark. This regional distribution suggested that particular strains might predominate in some areas. The extended phage patterns with the experimental phages did not give any retrospectively useful epidemiological information. It is proposed that in future phages 16 and 47A be used for specific investigations into the sources and relatedness of strains involved in small incidents.

Enzymuria in neonates receiving continuous intravenous infusion of gentamicin.

Urinary excretion of the tubular enzymes NAG and AAP was investigated during gentamicin treatment of 105 newborn infants. The values found for NAG and AAP show a significant positive correlation. The urinary excretion of NAG was on the average 92% higher during gentamicin treatment as compared with non-treatment periods in the same newborn infant (33 infants). The same tendency applied to AAP. Newborn infants receiving continuous intravenous infusion of gentamicin were not found to be at greater risk of nephrotoxicity than those receiving intermittent gentamicin treatment, using NAG and AAP as an index of nephrotoxicity. The changes in NAg and AAP within treatment periods were studied. During gentamicin treatment an insignificant average increase in the urinary excretion of NAG occurred, whereas a significant decrease was found during non-treatment periods. A significant negative correlation was found between urinary excretion of NAG and birth weight/gestational age. The long-term effect of the higher excretion of NAG and AAP in newborn and adult patients during aminoglycoside treatment is unknown.

Comparative analysis of two blood culture systems (Isolator and a 12-tube system) by cumulative differences in detection power at different times during incubation.

A lysis-centrifugation blood culture system (Isolator) and a conventional system (4 tubes of nutrient broth, 4 tubes of semisolid agar, and 4 tubes of thioglycollate agar) were compared after different lengths of incubation by cumulative scoring of differences in detection power. After the first half day of incubation, the Isolator system was already significantly faster in detecting isolates of clinical significance (15 vs. 4, P = 0.02). Maximum difference in first or only detection system was seen after two days of incubation and was based on an overall superior detection of Staphylococcus aureus (11 vs. 0, P = 0.001), and an earlier detection of Enterobacteriaceae (30 vs. 13, P = 0.01) in the Isolator system. On the contrary, the detection of Streptococcus pneumoniae was significantly inferior in the Isolator system (0 vs. 10, P = 0.002). The earlier finding of clinically significant microorganisms in the Isolator system certainly contributes to good patient-care. A drawback of the Isolator system was the finding of clinically insignificant coagulase-negative staphylococci in 11%, compared with 1% in the conventional system. This led to a considerable waste of time and materials in the laboratory. The comparison of the two blood culture systems, based on statistical analysis of cumulative differences in detection power, expressed as the earliest or only findings, gives the optimal information, and is in our opinion the clinically most relevant comparison.

Epidemics of Mycoplasma pneumoniae infection in Denmark from 1958 to 1974.

The study is a retrospective and prospective serological investigation of the incidence of Mycoplasma (M.) pneumoniae infection in Denmark during a 17-year period. By least square multiple regression analysis it was shown that four major outbreaks or epidemics had occurred which culminated at regular intervals of four and a half years. The study was based on cold agglutinin (CA) positive sera tested for antibodies to M. pneumoniae by an indirect immunofluorescence (IF) test, an indirect haemagglutination (IHA) test or a complement fixation (CF) test. By the criteria chosen for a positive test, the CF test was found to detect more cases than the IF or IHA tests. A shift during the study from the latter two tests to the CF test influenced the incidence, but not the periodicity of epidemics. The consequence of including only CA positive sera in the study was investigated.

24-h ambulatory blood pressure in 352 normal Danish subjects, related to age and gender.

UNLABELLED: The study was conducted to determine age and sex stratified normal values for 24-h ambulatory blood pressure. A sample of 352 healthy subjects (all white) were randomly selected from the community register and stratified by sex and age groups in decades from 20 to 79 years of age. Persons with a history of hypertension, cerebral apoplexy, diabetes, myocardial or renal disease, and who were taking blood pressure-influencing medication were excluded. Ambulatory blood pressure was recorded over 24 h, with measurements taken every 15 min from 07:00 to 22:59, and every 30 min from 23:00 to 6:59. Systolic blood pressure increased only slightly with age and was significantly higher in men than in women. The diastolic blood pressure increased only slightly with age in both sexes until the 50 to 59 years age group and declined thereafter. The diastolic blood pressure was not different for the two sexes. Both systolic and diastolic blood pressure were approximately 15% lower during the night regardless of age or sex. Ambulatory blood pressure during the daytime was on an average of 5 mm Hg lower than office blood pressure, but the mean difference between the two measurements increased with age. The variability of the difference also increased with age. IN CONCLUSION: Normal values for ambulatory blood pressure are presented in a randomly selected age- and gender-stratified population. Differences between office blood pressure and ambulatory blood pressure increased with age suggesting that the previously observed higher blood pressure seen in the elderly partly might be explained by a greater impact of white coat hypertension in older people.

Smoking related to 24-h ambulatory blood pressure and heart rate: a study in 352 normotensive Danish subjects.

This study shows the association between smoking and both office and ambulatory blood pressure. By means of stratification, a uniform number of subjects of both sexes and spanning 6 decades (aged 20 to 79 years) were recruited randomly from the local community register. A total of 352 subjects participated, including 161 smokers. Smokers (both sexes and all age groups summed), as compared with nonsmokers had statistically significant lower office blood pressure as follows (mean systolic +/- SED/mean diastolic +/- SED): (systolic and diastolic, -6.8 +/- 2.1/-3.9 +/- 1.3); day ambulatory blood pressure (diastolic, /-2.8 +/- 1.0); and night ambulatory blood pressure (systolic and diastolic, -4.2 +/- 1.8/-3.9 +/- 1.1). The intraperson variability of the day ambulatory blood pressure (as measured every 15 min) was identical for the smokers and the nonsmokers. Smokers were found to have a diminished "white coat" effect; this diminished white coat effect has not previously been described. The major white coat effect was seen in the older nonsmokers, whereas the diminished white coat effect was most pronounced in the older male smokers and in the younger female smokers. Smokers seem to have a diminished white coat effect, as well as a lower ambulatory blood pressure throughout the day (diastolic) and at night (systolic and diastolic). The similar intraperson variability found in the smokers' and nonsmokers' blood pressure further speaks for a consistently lower blood pressure in smokers as compared with nonsmokers.

The susceptibility of Plasmodium falciparum to sulfadoxine and pyrimethamine: correlation of in vivo and in vitro results.

In 1982, 2 of 14 Plasmodium falciparum infections acquired in East Africa and diagnosed in Copenhagen were resistant to treatment with sulfadoxine plus pyrimethamine (Fansidar), while in 1983, 6 of 18 were so. The in vivo tests were supplemented by determinations of drug concentrations in serum, and 4 isolates from in vivo-sensitive cases and 6 from in vivo-resistant cases were selected for in vivo tests. These were performed in ordinary RPMI 1640 medium and in a medium with physiological p-aminobenzoic acid and folic acid concentrations. Pharmacokinetic aberrations were found to be of possible importance in only 2 of the in vivo-resistant cases. In vitro susceptibility to sulfadoxine was found to be uniformly low in all isolates. Testing with a combination of sulfadoxine and pyrimethamine in the medium with physiological concentrations of cofactors probably reflects the in vivo situation most accurately, but in all but 1 of the isolates studied in vitro the in vivo susceptibility to Fansidar would be predicted by in vitro susceptibility to pyrimethamine in either medium. The concentration of p-aminobenzoic acid in serum, quantitated by high performance liquid chromatography, was found to be subject to wide variation, and this may have implications for in vitro testing.

Colonization priority among Staphylococcus aureus strains--correlation with phage-type.

We have studied the distribution of phage-type patterns among strains of Staphylococcus aureus isolated from patients in a burns unit. From 51 patients the same phage-type was isolated from succeeding swabs during the observation period. In 20 patients new types were introduced, but the original strain remained. In 23 patients the first strain was replaced by one other strain, in eight patients two or more. Strains of type 95 seemed to have a high colonization priority, whereas strains of group III had a low one. In 1986 phage-typing was performed on two or more S. aureus strains from the same patient, in 4561 instances. Recurrence of strains of the same phage-type pattern was demonstrated in 70% of the patients when the first and the fourth sample were compared. The "newer epidemic" strains of phage-type 95 and of the 94,96 complex had the highest percentage of recurrence (more than 80%) when adjacent samples were compared, and 68-69% when the first and the fourth sample were compared. The good colonization capacity of these strains might be one of the explanations why they occur frequently today although they are resistant only to penicillin.

Catheter-related Staphylococcus aureus infections.

Among 3394 patients with Staphylococcus aureus bacteraemia from the years 1986-89, 88 patients were found whose intravenous catheter and blood grew organisms of the same phage type. Strains of phage type 95 were more frequent among the patients with confirmed catheter-related bacteraemia than among other bacteraemia cases. Strains with particular phage-type patterns occurring with increasing frequency in Denmark during recent years also occurred with significantly higher frequencies among the confirmed catheter-related bacteraemias. No major differences in antibiotic resistance were observed. Patients with catheter-related bacteraemia had, in spite of a higher frequency of underlying diseases, a lower mortality compared with other bacteraemia patients, and endocarditis occurred less frequently (2% vs. 6%). Among 201 S. aureus isolates from catheters in 1988 only strains of group I occurred with increased frequency. The possible role of catheters as selection pressure on the S. aureus population is discussed.

Acute phase proteins and clinical synovitis activity in patients with rheumatoid arthritis.

A number of laboratory variables, including Hb., ESR and several phase proteins, fluctuated in concord with the clinical signs of synovitis activity in patients with rheumatoid arthritis during a controlled study of 3 disease-modifying anti-rheumatic drugs (DMARD). The correlation between laboratory variables and clinical synovitis was significant in a large patient population but the correlation coefficients were not of such magnitude that any of the laboratory variables reflected clinical synovitis activity in a reliable manner in the individual patients. In patients treated with azathioprine, the response of the Hb, (and consequently of the ESR), was reduced compared to patients given other DMARD. This phenomenon was caused by the bone marrow suppressing effect of azathioprine. However, the effect of azathioprine on the clinical synovitis activity did not differ from that of the 2 other drugs. Similar results were found by reviewing the literature about controlled trials of DMARD. In the present trial the clinical evaluation was performed under optimal conditions. In daily clinical practice the evaluations of the joints may be less than optimal since they may be performed by different rheumatologists with varying experience. Consequently, it may be difficult to do without the unreliable laboratory variables mentioned in the routine assessments of disease activity, unless the quality of routine evaluations of synovitis activity is improved considerably.

Influence of previous gold treatment and other patient variables on outcome of treatment with disease modifying anti-rheumatic drugs (DMARD) in patients with rheumatoid arthritis.

Based on a 2-year controlled double-blind trial of levamisole, penicillamine, and azathioprine (L, P, and A), a computer aided search for predictive factors of outcome was instituted. Already at month 4 several indicators of synovitis activity were able to discriminate between patients staying in the trial for 24 months and patients whose treatment was discontinued before that time. Patients who had previously received gold therapy responded less favourably to L, P, and A than those who had not received gold. This reduction of response was more pronounced in gold resistant patients than in patients whose gold treatment had been discontinued for other reasons. The only phase protein (of several) with a predictive value was haptoglobin. If, after 4 months of treatment, haptoglobin did not normalize, this finding indicated a lack of response to treatment or a deterioration of synovitis activity during the following 4 months. The response to treatment was not influenced by HLA-types, sex, age, or clinical synovitis, disease duration, functional or anatomical aberrations at the start of treatment. The shape of the response curve as reflected by means of monthly measurements of serum-albumin and ESR was not related to disease duration, HLA-types, or previous gold treatment.

[Treatment of gonorrhea at the Venereal Disease Clinic in Copenhagen in 1985. A study of the efficacy of standardized treatment compared with the sensitivity of Gonococcus to penicillin in vitro]. / Behandling af gonoré på Københavns Venereaklinik i 1985. En undersøgelse af effekten af standardbehandling sammenholdt med gonokokkernes følsomhed for penicillin in vitro.

In 1985, a total of 1,140 patients (907 men and 503 women) were treated for gonorrhoea at the Copenhagen County Outpatients Clinic at Rudolph Berghs Hospital. Failure of the standard treatment regimen (pivampicillin plus probenecid; patients stating penicillin allergy received a Sulfotrim-regimen) occurred in 79 (5.6%). A significant association between the in vitro susceptibility to penicillin and the efficacy of the standard treatment regimen was found; among 1,139 patients with uncomplicated urogenital gonorrhoea caused by penicillin susceptible gonococci, the failure rate was low (0.4-1.9%); the failure rate was high in patients infected with penicillin resistant gonococci. Continued surveillance of the in vitro antimicrobial susceptibility of gonococci is recommended as a basis for the choice of standard treatment regimen.

[Round-the-clock blood pressure measurements in 352 persons--a reference material]. / 24 timers ambulant blodtryk hos 352 personer--et referencemateriale.

The aim of this study was to establish reference values for 24-hour ambulatory blood pressure in a Danish population stratified for gender and age in the decades from 20 to 79 years of age. A sample of 352 persons, 179 men and 173 women randomly selected from the local community register, age 20-79 years underwent 24-h ambulatory blood pressure monitoring. For men age < 50 daytime ambulatory blood pressure (median) was 125/79 mmHg and night time was 106/65 mmHg, for women the respective pressures were 113/77 mmHg and 97/64 mmHg. For men age > or = 50 daytime ambulatory blood pressure was 133/83 mmHg and night time was 124/86 mmHg, for women the respective pressures were 122/83 mmHg and 105/65 mmHg. Presently, we can only relate cardiovascular risk to clinic blood pressure. Therefore we have calculated corresponding ambulatory blood pressure values to WHO's upper limit 160/90 mmHg for normal blood pressure in the clinic and found 154/87 mmHg for daytime and 134/74 mmHg at night. For a clinic pressure of 95 mmHg the corresponding daytime value was 91 mmHg, for 100 mmHg it was 95 mmHg.