Reference ranges and cutoff levels of pneumococcal antibody global serum assays (IgG and IgG2) and specific antibodies in healthy children and adults.

Pneumococcal antibodies represent the acquisition of natural immunity. Determination of pneumococcal antibodies is an important screening tool for immunodeficiencies. Our study generated reference ranges and cutoff levels for pneumococcal antibody global serum assays correlated to a specific pneumococcal antibody ELISA. Specific pneumococcal antibody levels were measured from 457 children undergoing elective surgery and 46 healthy adult volunteers (88 with previous pneumococcal immunization from both groups), 22 severe immunodeficient subjects with ataxia telangiectasia (A-T, negative controls), and age-matched 36 healthy allergic asthmatics. We determined a representative panel of serotype-specific pneumococcal antibodies (serotype 4, 5, 6B, 7F, 14, 18C, 19F, 23F) by ELISA and global pneumococcal IgG and IgG2 antibodies by EIA. In vaccine-naïve healthy subjects, initial pneumococcal IgG geometric mean concentrations of 13.1 µg/ml were low in the first year of life and increased over the time, reaching adult levels (70.5 µg/ml) at age 8-12 years. In parallel, IgG2 antibodies increased from 20.7 % (0.5-1 year old) to adult proportions (>30 %) in preschoolers. Correlation between the pneumococcal IgG screening assay and specific pneumococcal antibody levels was acceptable (Pearson's coefficient r = 0.4455; p = 0.001). Cutoff levels showed high sensitivity, whereas specificity was high to moderate calculated from correlations with the specific ELISA. We provide reference ranges and cutoff levels for the interpretation of specific antibody determinations in the clinical setting. The global pneumococcal IgG/IgG2 assay is a suitable screening tool and correlates with the ELISA serotype-specific pneumococcal antibodies. However, results below our cutoff values should be re-evaluated by serotype-specific ELISA testing.

Biological inactivation of pBR322 plasmid DNA by enzyme- and radiation-induced single-strand damage under various conditions.

The influence of three different kinds of single-strand breaks (ssb) on the biological activity of plasmid DNA (pBR322) was studied. The single-strand breaks were produced either by gamma-irradiation (together with base and sugar damage) or by DNase I digestion which introduced ligatable ssb. Non-ligatable ssb--single-strand gaps of three nucleotides in length--were generated in the nicked DNA by exonuclease III treatment. The biological activity (N/N(o)) of this damaged DNA was assessed in vivo by transformation of E. coli (CMK) repair wild-type cells. The activity of the enzymes of E. coli was studied in vitro by incubation in a protein extract of E. coli making use of an in vitro assay introduced earlier, which makes it possible to distinguish between enzymatic degradation (dsb formation) and repair of damaged plasmid DNA. The biological activity (D37) of DNA with non-ligatable ssb, as determined by electrotransformation, was about 56% lower than that of DNA with ligatable ssb. The biological activity of enzymatically damaged DNA is greater in calcium-treated cells than in electroporated cells. It is proposed that this is due to a calcium-dependent inhibition of nucleases. In contrast to the enzymatically damaged DNA, with gamma-radiation-damaged DNA a calcium-dependent increase in survival was not observed. Therefore, calcium-dependent nucleases do not play a role in the repair of damage produced by gamma-irradiation. The enzyme activity data show that the single-strand damages are either converted into dsb or repaired. A comparison of the efficiency of dsb formation in the extract for two of the single-strand damages is presented. The efficiency depends on the kind of damage and on the presence of cofactors, especially ATP and dNTPs.

Effects of adhesins from mannose-resistant Escherichia coli on mediator release from human lymphocytes, monocytes, and basophils and from polymorphonuclear granulocytes.

We investigated the role of Escherichia coli expressing mannose-resistant hemagglutination and adhesins with regard to the induction of leukotrienes from a suspension of human lymphocytes, monocytes, and basophils (LMBs) compared with human polymorphonuclear granulocytes (PMNs). Genetically cloned E. coli strains expressing various types of mannose-resistant hemagglutination (MRH+) were phagocytosed to a higher degree by monocytes than the nonadherent E. coli strain. The various strains differed in their capacity to induce a chemiluminescence response, which showed the same pattern for LMBs and PMNs. Stimulation of LMBs with bacteria alone, unlike granulocytes, did not activate the cells for the release of leukotrienes. However, preincubation of LMBs with bacteria decreased subsequent leukotriene formation when the cells were stimulated with calcium ionophore. The inhibitory effect was dependent on the concentration of bacteria used for preincubation as well as on the preincubation temperature. The various bacterial strains differed in inhibitory potency for mediator release. Preincubation of LMBs with zymosan, opsonized zymosan, the bacterial peptide FMLP, and peptidoglycan had no inhibitory effect or even increased subsequent leukotriene formation. Opsonized bacteria were far less inhibitory than nonopsonized bacteria. In contrast to human LMBs, preincubation of human PMNs with mannose-resistant bacteria led to increased leukotriene B4 generation and reduced w-oxidation of leukotriene B4. Our data suggest that phagocytes (neutrophils, monocytes) respond in a different way for leukotriene formation after interaction with mannose-resistant E. coli.