Methodologic problems in establishing normal values for IgG subclass concentrations in a pediatric population; comparison of radial immunodiffusion and ELISA methods.

The aim of this study was to establish an enzyme-linked immunosorbent assay (ELISA) to measure IgG subclasses by means of monoclonal antibodies. The distribution of IgG subclass protein concentrations in sera from 227 healthy Danish children and 90 adults was measured. Furthermore, this newly established ELISA was compared with different assay systems for determination of IgG subclasses: two radial immunodiffusion methods (RID), one using polyclonal and one using monoclonal antibodies, as well as a commercially available ELISA kit. There was good agreement of results obtained by the different methods of measuring IgG3 and IgG4 concentrations. There was good correlation between results obtained by both RID methods. Despite good correlation between the assays, the ELISA kit showed higher levels of IgG1 in all investigated sera, and the ELISA kit showed no correlation with the other methods, when IgG2 was measured. Analysis of the normal ranges measured by ELISA developed in our laboratory and by RID with polyclonal antibodies showed that the levels obtained by RID were higher than those obtained by our ELISA in sera with low levels of both IgG1 and IgG2, and lower in sera with high concentrations of these two immunoglobulins. Our results emphasize the importance of establishing age-related normal limits for any novel assay measuring IgG subclass concentrations.

beta-lactamases in Shigella.

The occurrence of resistance and production of beta-lactamases was investigated in 60 Shigella strains. Ampicillin resistance was found in 28 (47%) of the isolates, the resistance being more frequent in Sh. flexneri than in Sh. sonnei. All strains were susceptible to cefotaxime, mecillinam, and ciprofloxacin. The beta-lactamases produced by Shigella were similar to TEM-1, OXA-1, or the low-level chromosomally mediated cephalosporinase produced by Escherichia coli. The beta-lactamases produced by Sh. flexneri were most often the OXA-1-like enzymes.

Screening method for beta-lactamase substrate profiles.

A disc diffusion method, based on the idea of Klundert, for screening of substrate profiles of beta-lactamases was developed in order to perform epidemiological studies. The method was tested against 30 different reference beta-lactamases and 59 clinical isolates known to produce TEM-1, SHV-1 and BRO-1. The reproducibility and discriminating power of the disc diffusion method made it possible to differentiate between eight types of substrate profiles for the 30 reference beta-lactamases and to differentiate between TEM-1, SHV-1 and BRO-1 from clinical isolates. In combination with analytical isoelectric focusing the disc diffusion method gives a reliable identification of beta-lactamases.

Polyacrylamide gel electrophoresis analysis of lipopolysaccharide from Pseudomonas aeruginosa growing planktonically and as biofilm.

Lipopolysaccharide (LPS, endotoxin) was extracted from biofilm and planktonically grown monoagglutinable (1118) and polyagglutinable (258 and 15703) strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients with chronic pulmonary infections. Analysis by polyacrylamide gel electrophoresis (PAGE) followed by immune-detection of LPS fractions showed an S-form appearance of strain 1118 and 258 with three distinct clusters of high molecular weight bands, whereas 15703 appeared semi-rough. LPS of semi-rough cells grown planktonically and as biofilm showed a very similar PAGE pattern; however, the core/lipid A R-LPS fraction was more prominent in biofilm-LPS than in planktonic-LPS extracted from the S-form bacteria (1118 and 258). The apparent change in LPS sub-unit components of the bacteria when grown as biofilm may reflect changes in the outer membrane structure that contribute to the altered physico-chemical properties of biofilm bacteria in foreign-device associated infections and chronic P. aeruginosa lung infection in cystic fibrosis patients.

Determination of IgG subclass antibodies to Pseudomonas aeruginosa outer membrane proteins in cystic fibrosis lung infection using immunoblotting and enzyme-linked immunosorbent assay.

IgG subclass antibodies to Pseudomonas aeruginosa outer membrane proteins (OMP) were investigated in serum from cystic fibrosis (CF) patients by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Fifteen patients (eight in good and seven in poor clinical condition) have been followed for an average of 13 years with multiple serum samples covering the preinfection, and early and late stages of chronic infection. Laser-scanning densitometry of photographs taken from immunoblots was used to quantify antibody level and compare with ELISA titres. The earliest anti-OMP antibodies to appear were of the IgG1 subclass. There was no significant difference in IgG subclass antibody levels to OMPs between patients in relatively good and poor clinical condition. Data presented indicate a high positive correlation among measurements of IgG subclass antibodies to P. aeruginosa OMPs using ELISA and immunoblotting.

Morphology of Pseudomonas aeruginosa phages from the sputum of cystic fibrosis patients and from the phage typing set. An electron microscopy study.

Sixteen phage suspensions isolated from the sputum of sixteen cystic fibrosis patients and five phages from the present phage typing set were studied by electron microscopy. All sputum samples contained at least one type of bacteriophage (range: 1-4) which could be classified by the morphology and dimensions of the virion. All phages isolated from sputum as well as the four typing phages were tailed phages. The clinical phages belonged either to the Myoviridae, Siphoviridae or Podoviridae family. The four typing phages belonged to the Myoviridae family. The detection of the presence of tailed phages in sputum further supports the prevailing theory that phages play a role in the phenotypical change of Pseudomonas aeruginosa during the chronic lung infection of cystic fibrosis patients, since only tailed phages are known to be temporate and thus mediate transduction and conversion.

Immune dysfunction in multiple myeloma. Reduced natural killer cell activity and increased levels of soluble interleukin-2 receptors.

Multiple myeloma (MM) is characterized by an increased susceptibility to infections and to other malignancies. Selected related immune functions were studied. Spontaneous and interleukin-2-stimulated natural killer (NK) cell activities were normal in 19 patients with MM compared with 62 controls. In contrast, interferon-stimulated NK cells had a significantly lower increase in activity in MM than in controls. The normal improvement in lytic NK cell activity after addition of indomethacin to the mononuclear cell cultures (to inhibit prostaglandin-mediated suppression) was not observed in cultures from MM patients. As reported for other lymphoproliferative disorders, the levels of soluble interleukin-2 receptors in serum were significantly higher in MM (600 U/ml median value) compared with controls (317 U/ml median value), P less than 0.0001, and the concentration of interleukin-2 receptors was significantly correlated with the concentration of monoclonal immunoglobulin in serum. Blood monocyte chemotactic responsiveness was significantly lower in MM patients with both zymosan-activated serum and f-Met-Leu-Phe as cytotaxins, suggesting reduced ability to accumulate at inflammatory foci. In contrast, release of reactive oxygen radicals, believed to be associated with the killing ability of monocytes, was normal after in vitro stimulation.

In vitro cytotoxic effect of alpha-hemolytic Escherichia coli on human blood granulocytes. Inhibition by alpha-hemolysin antibody.

The influence of alpha-hemolysin antibody on the in vitro cytotoxic effect of alpha-hemolytic Escherichia coli bacteria and culture filtrates was investigated. Damage to human blood granulocytes was quantified by measuring the release of chromium 51 from labelled cells in the presence of whole or fractionated plasma containing alpha-hemolysin antibody. Anti-alpha-hemolysin activity was found exclusively in the IgG fraction of plasma. Human plasma contained "natural" alpha-hemolysin antibody to various titers. Vaccination of rabbits resulted in only modest rises in alpha-hemolysin antibody titers. The cytotoxic effect of alpha-hemolytic E. coli bacteria as well as that of bacterial culture filtrates was reduced in a titer-dependent way in the presence of plasma containing alpha-hemolysin antibody. These results indicate that the cytotoxic effect of alpha-hemolytic E. coli is inhibited by alpha-hemolysin antibody.

Intravenous immunoglobulin: a single-blind trial in children with Lennox-Gastaut syndrome.

The antiepileptic effect of intravenous immunoglobulin (Sandoglobulin, Sandoz) was investigated in Lennox-Gastaut syndrome by an add-on, placebo-controlled, single-blind trial. Ten patients, aged 4-14 years, with insufficient response to conventional anticonvulsive therapy received placebo and Sandoglobulin 400 mg/kg two times each with an interval of two weeks. The washout period was four weeks and the total observation period 14 weeks, during which parents daily registered number and type of seizures. EEG, in vitro lymphocyte transformation tests and concentrations of immunoglobulins including IgG subclasses were evaluated before and after active treatment. Two children showed an immediate reduction in their high-frequency and invariable seizure activity from 42% to 100% and a less abnormal EEG. In addition, general well-being and intellectual performance was improved. The strongest response was observed in one child with a concomitant finding of a low level of IgG2, the only abnormal immunologic test in this study. The remaining 8 children, who had either a high or a low but variable seizure frequency showed no immediate change as EEG and their general condition was unaffected. We conclude that intravenous immunoglobulin had an immediate and pronounced effect on break-through seizure activity and a simultaneous neurophysiologic effect in 20% of our patients with Lennox-Gastaut syndrome. The effect was not confined to patients with immunologic abnormalities.

Studies on beta-lactamases from Escherichia coli isolated from urinary tract infections.

The beta-lactamase types present in 75 ampicillin and carbenicillin resistant E. coli were characterized using isoelectric focusing (IEF). The strains were isolated from patients with urinary tract infections from two geographically different areas of Denmark: 38 strains from Copenhagen and 37 strains from North Jutland. For 19 of the strains from Copenhagen and 18 of the strains from North Jutland, their beta-lactamase activity against nitrocefin and ampicillin, carbenicillin, benzylpenicillin, cloxacillin and cephaloridine was examined by a micro-iodometric and an UV-spectrophotometric assay. The strains from Copenhagen showed greater activity (p less than 0.001) against nitrocefin than the strains from North Jutland. The rate of hydrolysis of ampicillin was greater for the strains from Copenhagen than for the strains from North Jutland. Ninety-three per cent of the strains produced plasmid-mediated beta-lactamases, of which the most prevalent, TEM-1, was produced by 97 per cent of these strains, and OXA-1 by 3 per cent.

The effect of ranitidine on cellular immunity in patients with multiple myeloma.

Multiple myeloma is characterized by an increased susceptibility to infections and to other malignancies. In a double-blind, placebo-controlled study the potential impact of immunomodulation by ranitidine was studied in 20 patients with multiple myeloma. Three patients were untreated, while 17 after previous cytotoxic therapy were in a stable phase of their disease. All were without clinical signs of infections and at that time had not been treated with other immunomodulating agents. The patients were randomized to oral ranitidine 300 mg twice a day for 21 days or placebo, and several immunological parameters related to multiple myeloma were studied. The blood monocyte chemotactic response was improved in patients treated with ranitidine, and superoxide anion production increased from 2.02 nmol/min to 3.86 nmol/min (median values), while it was unchanged in patients given placebo (2.19-2.25 nmol/min) (P less than 0.005 between groups). Among ranitidine-treated patients spontaneous NK cell activity was unchanged, while in vitro interleukin-2- and interferon-alpha-stimulated NK cell activity decreased (P less than 0.03, respectively). As production of oxygen radicals constitutes an important mechanism of monocyte killing activity against microorganisms and probably against malignant cells, it is suggested that ranitidine may be of beneficial impact in the treatment of multiple myeloma.

Complement and immunoglobulin studies in 15 cases of chronic meningococcemia: properdin deficiency and hypoimmunoglobulinemia.

The purpose of this study was to investigate whether patients with chronic meningococcemia have abnormalities in their humoral immune system. The alternative and classical complement system, the levels of IgA, IgG and IgM, as well as IgG subclasses were studied in 15 individuals who had recovered from chronic meningococcemia. We found one individual with complete deficiency of properdin, a component of the alternative complement pathway. In the other patients, the complement system was normal. The mean plasma IgG concentration was significantly below normal in the patient group, while the mean values of IgA, IgM and the IgG subclasses were normal. Two individuals, however, had low IgG2 and IgG4 levels. We conclude that properdin deficiency and reduced plasma IgG levels may predispose to chronic meningococcal disease, but that the majority of patients with chronic meningococcemia have a normal humoral immune system.

Complement-fixing properties of human IgA antibodies. Alternative pathway complement activation by plastic-bound, but not specific antigen-bound, IgA.

The complement-fixing properties of human IgA antibodies bound to specific antigen, or coated directly on plastic surfaces, were examined in comparison with those of IgG antibodies. Use was made of antigen-binding (anti-staphylococcal alpha-toxin) IgA and IgG monoclonal antibodies and normal polyclonal IgA and IgG, purified greater than 99.9% by avoidance of denaturing processes. Complement-fixation ELISA was used, with a high density of biotin-conjugated staphylococcal alpha-toxin bound to avidin-coated plates for the efficient capture of antibodies, and conditions were adjusted for the assessment of classical and alternative pathways of complement activation. Although IgA coated directly on plastic surfaces activated the alternative complement pathway in a dose-dependent manner, IgA antibodies bound to antigen failed to fix complement by either classical or alternative pathways. In contrast, IgG antibodies, either bound to antigen or coated directly on plastic, activated complement mainly by the classical pathway. It was concluded that the complexation of IgA antibodies with antigen is insufficient to elicit complement activation: rather a degree of denaturation seems to play a part in the expression of alternative complement pathway-activating properties by IgA.

Increased IgG2 and IgG3 concentration is associated with advanced Pseudomonas aeruginosa infection and poor pulmonary function in cystic fibrosis.

The concentrations of IgG subclass immunoglobulins were determined by radial immunodiffusion in serum from 126 patients with cystic fibrosis (CF). The results were compared to values from age-matched healthy children and adults and correlated to patients age, duration of chronic Pseudomonas aeruginosa infection and lung function parameters. Fifty-two percent of the patients had an elevated concentration of at least one of the IgG subclasses; IgG1 28%, IgG2 16%, IgG3 18% and IgG4 48%. There was significant correlation between elevated serum levels of IgG2, and to a lesser extent IgG3, with decreased lung function (for FEV1; p = 0.0001, and p = 0.001 respectively) and high levels of antipseudomonas precipitins (p = 0.008, and p = 0.002). A similar correlation was not found for IgG1 and IgG4. IgG subclasses vary in their ability to promote phagocytosis and to activate complement and it is possible that individual differences in the IgG subclass pattern could explain the variable course of this disease.

IgG subclass concentrations in sera from 200 normal adults and IgG subclass determination of 106 myeloma proteins: an interlaboratory study.

The IgG subclass protein concentrations in sera from 200 normal subjects were determined independently in two laboratories, using the same technique (radial immunodiffusion), the same subclass-specific antibodies and the same calibrator. The coefficients of correlation (rs) between IgG subclass concentrations determined in the two laboratories were 0.487, 0.883, 0.928 and 0.926 for IgG1, IgG2, IgG3 and IgG4, respectively (p less than 0.0005 in all four cases). By the chi-square test for goodness of fit, the frequency distributions observed in the two laboratories were found to differ significantly for all four subclasses (p less than 0.01). In one laboratory, the distributions of IgG1 and IgG2 were not significantly different from normal distributions, whereas the distributions of IgG3 and IgG4 deviated significantly. In the other laboratory, all four subclass distributions were significantly different from normal distributions. In the first laboratory, the IgG2 concentrations were log-normal distributed, whereas in the second, IgG1, IgG2 and IgG4 concentrations were log-normal distributed. We conclude that use of identical reagents does not ensure identical frequency distributions. This finding emphasizes the need for standardization of the measurement technique too. Furthermore, we argue that at present, intralaboratory reference intervals for IgG subclass protein concentrations are necessary. The reference intervals should be based on non-parametric statistics. The subclass of 106 monoclonal IgG proteins, which were demonstrated by agarose gel electrophoresis, was identified by RID for 89 samples. The subclass of the remaining 16 M-components was readily determined by qualitative immunoelectrophoretic analysis using the same subclass-specific antibodies.

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.

Comparison of resistance types in Enterobacter cloacae 1977 and 1985.

A collection of Enterobacter cloacae strains from Odense University Hospital from 1977 were compared with a collection from 1985 as regards acquired resistance traits. Among the strains with carbenicillin (Ca) resistance, the number of multiresistant strains decreased while the number with sole Ca-resistance increased. In 1977, a high proportion of the Ca-resistant (Ca-r) strains had plasmid-mediated beta-lactam resistance, but in 1985 the Ca-r strains were completely dominated by organisms with elevated amounts of chromosomally-mediated beta-lactamase. The latter, but not the former, strains were resistant to the newer cephalosporins (e.g. cefotaxime (Ctax)). The consumption of Ctax and cefuroxime increased from 0 kg in 1977 to 7.0 kg in 1985. It is therefore probable that this increase was the cause of the change in occurrence of the resistance types. Ninety-one % of the Ca-r strains were isolated from urinary samples in 1977. The percentage was only 31 in 1985. This change, concomitant with the increase in Ctax-r strains, can probably be explained by the better conditions for selection of Ctax-r mutants, producing greater amounts of chromosomal beta-lactamase, in wounds and respiratory tract than in urine.

Circulating IgG from patients with primary Sjögren's syndrome deposited in the epidermis of normal human skin transplanted to athymic nude mice.

Sera from 7 patients with primary Sjögren's syndrome and from two control persons were administered intraperitoneally to athymic nude mice transplanted with normal human skin. Seven days after transfer of serum from 5 of the patients, intra-epidermal IgG1 and IgG3 deposits were demonstrated in the skin grafts by immunofluorescence. The deposits closely resembled in vivo deposits found in the skin of these patients. No correlation was found between the presence of epidermal deposits and levels of IgG1 and IgG3 in serum. No IgG deposits were found in skin grafts on animals given control serum, and neither could human IgG be detected in mouse skin adjacent to the grafts. Epidermal deposits of human-IgM, -IgA, -fibrinogen, -C3c and mouse-Ig were not demonstrated in biopsies from grafts or mouse skin. The results support the hypothesis that epidermal in vivo deposits of IgG in patients with primary Sjögren's syndrome are the result of Fc-receptor-mediated binding to epidermal cells.

Pathogenicity and persistence of pleural effusion disease virus isolates in rabbits.

Nine isolates of pleural effusion disease agent or virus (PEDV) from treponema-infected rabbits in various countries were examined for pathogenicity and persistence in rabbits. The isolates showed a wide range of pathogenicity and were categorized into three groups according to the severity of the acute infection. Group 1 comprised isolates causing more than 50% mortality, group 2 isolates causing mortality below 50%, while group 3 comprised isolates causing almost subclinical infections. The range between group 1 and group 3 was similar to that observed with virulent and avirulent progeny of the original PEDV isolate. Infection by each of the nine isolates resulted in a chronic low level viraemia which persisted for up to 2 years or more. Viral progeny of pathogenic isolates obtained in serum after the 2nd month of infection failed to induce clinical disease on rabbit inoculation. The chronic, subclinical infection was associated with a moderate, continued increase in serum IgG, but circulating immune complexes could not be demonstrated. Two years after infection slight histopathological changes were present in lymph nodes, spleen, liver, heart and lung. Evidence of immune complex disease could not be demonstrated.

Retained antigen-binding activity of Fab alpha fragments of human monoclonal immunoglobulin A1 (IgA1) cleaved by IgA1 protease.

Immunoglobulin A1 (IgA1) proteases may be important virulence factors of certain bacteria involved in the pathogenesis of meningitis, gonorrhea, destructive periodontal diseases, and some other infections affecting mucosal membranes. This study evaluated the antigen-binding activity of free Fab alpha fragments released from human myeloma IgA1 by IgA1 protease from Haemophilus influenzae. Six myeloma proteins with antibody activity against streptolysin O, alpha-staphylolysin, or streptococcal hyaluronidase were used. Complete cleavage of the IgA1 myeloma proteins in the hinge region of the heavy chain did not affect their antigen-binding capacity. The titers of neutralizing activity associated with free Fab alpha fragments were not significantly different from those of the intact IgA1 proteins. The retained antigen-binding capacity of cleaved IgA1 is an important factor in the understanding of how IgA1 proteases may interfere with the immune protection of mucosal membranes.