Paediatric reference values for the peripheral T cell compartment.

Immunophenotyping of blood lymphocyte subpopulations is an important tool in the diagnosis of immunological and haematological diseases. Paediatric age-matched reference values have been determined for the major lymphocyte populations, but reliable reference values for the more recently described T lymphocyte subpopulations, like different types of memory T lymphocytes, recent thymic emigrants, regulatory T cells and CXCR5(+) helper T lymphocytes, are not sufficiently available yet. We determined reference values for the absolute and relative sizes of T lymphocyte subpopulations in healthy children using the lysed whole blood method, which is most often used in diagnostic procedures. When the absolute numbers of some or all T lymphocyte subpopulations fall outside these reference ranges, this may indicate disease. The reference values show the course of T lymphocyte development in healthy children. Absolute T lymphocyte numbers increase 1.4-fold during the first months of life, and after 9-15 months, they decrease threefold to adult values; this is mainly caused by the expansion of recent thymic emigrants and naive cells. Helper and cytotoxic T lymphocytes show the same pattern. Regulatory T cells increase in the first 5 months of life and then gradually decrease to adult values, although the absolute numbers remain small. The relative number of CXCR5(+) cells within the CD4(+) CD45RO(+) T lymphocytes increases during the first 6 months of life and then remains more or less stable around 20%.

Development of lymphocyte subpopulations in preterm infants.

In preterm neonates the immune system is thought to be less developed at birth, but very little is known about the actual size of lymphocyte subpopulations, and even less about the maturation of these subpopulations during the first months after a premature birth. To evaluate the development of lymphocyte subpopulations in preterm infants during the first 3 months after birth, we performed a prospective longitudinal study in two hospitals in the Netherlands. Preterm neonates (n = 38) of all post-menstrual ages were included and blood samples were taken from cord blood, and at 1 week, 6 weeks, and 3 months. Lymphocyte subpopulations were measured by four-colour flow cytometry. The data were compared with follow-up data obtained in healthy term neonates (n = 8), and with single samples from school age children (n = 5) and adults (n = 5). Overall, we found a similar pattern of post-natal development of lymphocyte subpopulations in the term and preterm infants. Both B lymphocytes and helper and cytotoxic T lymphocytes mainly consist of naive cells at birth and during the 3 months of follow-up in all neonatal age groups. So, the preterm immune system seems to be able to generate an outburst of naive T and B lymphocytes from the thymus and bone marrow within the same time span after the start of post-natal antigenic stimulation from the environment as the term immune system, but, with lower post-menstrual age, the absolute counts of naive helper T lymphocytes are lower.

Age-matched reference values for B-lymphocyte subpopulations and CVID classifications in children.

Age-matched reference values are generally presented with 5th and 95th percentiles as 'normal' reference range. However, they are mostly determined in relatively small groups, which renders this presentation inaccurate. We determined reference values for B-lymphocyte subpopulations in healthy children with the statistical method of tolerance intervals that deals far better with the relatively small numbers tested, and compared these to the cut-off values used in the currently used EUROclass classification for common variable immunodeficiency disorders (CVID) in children. CVID is a heterogeneous group of primary immunodeficiency diseases characterized by low serum immunoglobulin levels and inadequate response to vaccination. Disease-modifying heterozygous amino acid substitutions in TACI are found in around ±10% of CVID patients. Interestingly, we found that age is the primary determinant of TACI-expression on B-lymphocytes, independent of switched memory B-lymphocyte numbers. Immunophenotyping of B-lymphocyte subpopulations is increasingly used to classify patients with CVID into subgroups with different clinical prognosis according to the composition of their B-lymphocyte compartment. These classifications were mainly developed with data obtained in adults. Because of the maturing paediatric immune system, they may not be equally applicable in children: our and other age-matched reference values show great changes in the composition of the B-lymphocyte compartment during development. Although the greatest changes in B-lymphocyte subpopulations occur below the age of 2 years, when the diagnosis of CVID cannot yet be made, it is likely that a classification developed in adults cannot be used to classify the prognosis of children.

Isolation and chemical analysis of 7-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-L-glycero-D-manno-heptose as a constituent of the lipopolysaccharides of the UDP-galactose epimerase-less mutant J-5 of Escherichia coli and Vibrio cholerae.

Methyl 7-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-L-glycero-D-manno-hepto+ ++ pyranoside (1) was released from the lipopolysaccharide of the UDP-galactose epimerase-less mutant J-5 of Escherichia coli by methanolysis and isolated by high-voltage paper electrophoresis. Its chemical structure was determined by chemical analysis, deamination with nitrous acid, g.1.c.-m.s., and 1H- and 13C-n.m.r. spectroscopy performed on its acetylated derivative. The disaccharide moiety of 1 was also detected in lipopolysaccharides of Vibrio cholerae.

Tumor necrosis factor and interleukin-6 in critical illness polyneuromyopathy.

Critical illness polyneuromyopathy (CIPN) occurs in critically ill patients on artificial respiration. The pathophysiology of this disease is unknown. Because of the strong association with sepsis, the levels of cytokines, TNF and IL-6 were measured several times daily in patients having CIPN and in a control group of critically ill patients without CIPN. The diagnosis of CIPN was made on clinical criteria. Patients with CIPN had no significantly elevated levels of TNF or IL-6 as compared to controls.

Characterization of anti-core glycolipid monoclonal antibodies with chemically defined lipopolysaccharides.

Five anti-core glycolipid monoclonal antibodies (MAb) (four against Escherichia coli J5 lipopolysaccharide [LPS] and one against the Re core glycolipid of Salmonella typhimurium) were characterized using LPS from several rough and smooth strains and derivatives of E. coli J5 LPS, obtained by N acetylation and hydrolysis. The MAb against E. coli J5 were not only weakly cross-reactive with clinical isolates, whereas the anti-Re MAb was highly cross-reactive. The MAb differed in their reaction pattern with E. coli J5 LPS. MAb 4-7B5 (immunoglobulin M) and MAb 4-6A1 (immunoglobulin G1) cross-reacted with LPS of Salmonella minnesota R5 and S. typhimurium Ra and Rc and little with Re and lipid A. The dominant binding site of these MAb was located in the glucose-heptose-heptose region and was independent of phosphate substitution. The MAb 4-9A1 reacted with the terminal part of the core region (glucose-heptose) and was dependent on phosphate substitution of the LPS. The MAb BA7 (immunoglobulin G3) was E. coli J5 LPS specific and reacted with the glucosaminyl-heptose disaccharide. Antibody 8-2C1 was directed against the common parts of LPS, 3-deoxy-D-manno-octulosonic acid, and lipid A, which are not (or only weakly) recognized by the four anti-J5 LPS MAb. Thus, MAb that are not cross-reactive can be directed against at least three different antigenic determinants present on the core oligosaccharide of E. coli J5 LPS.

Complement activating and opsonic capacity of monoclonal antibodies raised against Escherichia coli O111 and its rough mutant J5.

Six monoclonal antibodies raised against Escherichia coli O111 and against its rough mutant J5 (chemotype Rc) were studied. One IgG2A, one IgM anti-J5, and one IgG2A anti-O111 monoclonal antibody did not bind to lipopolysaccharides of the homologous strain, but cross-reacted with heterologous gram-negative rods in an enzyme-linked immunosorbent assay. These three monoclonal antibodies activated complement when incubated with homologous or heterologous strains, but were opsonic neither in the presence nor in the absence of complement. The other three monoclonal antibodies were directed against lipopolysaccharide of the homologous strain, but showed no cross-reactivity. The IgG3 and one IgM anti-J5 monoclonal antibodies activated complement and were opsonic only in the presence of complement. The IgM anti-O111 monoclonal antibody activated complement and was opsonic both in the presence and absence of complement. Thus, the outcome of the interaction between bacteria, antibodies, and complement is influenced primarily by whether antibodies are directed against lipopolysaccharides or against other cell wall components.

Cross-reactivity of monoclonal antibodies against lipopolysaccharides of gram-negative bacteria.

Monoclonal antibodies were produced against Escherichia coli O111, Escherichia coli J5, and the rough (R) mutant of Salmonella typhimurium M206, and tested by enzyme-linked immunosorbent assay against lipopolysaccharides of several gram-negative strains. The monoclonal antibodies were also identified with an immunoblotting assay. Anti-Escherichia coli O111 monoclonal antibodies reacted only with homologous O antigens. Anti-J5 monoclonal antibodies cross-reacted with core lipopolysaccharide, especially with Rc lipopolysaccharide. IgM anti-J5 monoclonal antibodies showed more extensive cross-reactivity than IgG3 monoclonal antibodies. Anti-Re monoclonal antibodies cross-reacted weakly with all rough lipopolysaccharide tested. Thus, the varying specificity of these monoclonal antibodies seems to indicate that the core regions in the lipopolysaccharides of various gram-negative bacteria are not similar.

Detection of antibodies against lipopolysaccharides of Escherichia coli and Salmonella R and S strains by immunoblotting.

Antisera raised against several smooth and rough strains of Escherichia coli and Salmonella typhimurium were tested against lipopolysaccharides (LPS) of homologous and heterologous strains. The LPS were separated by sodium dodecyl sulfate-gel electrophoresis, transferred to nitrocellulose paper, and overlaid with antisera. The results showed that antisera raised against smooth strains reacted with high- as well as low-molecular-weight bands of their corresponding LPS and showed very few cross-reactions. Anti-E. coli J5 antiserum cross-reacted with few strains in the core region. But, anti-S. typhimurium Ra antiserum cross-reacted with many more strains. When these sera were absorbed with either the homologous- or a heterologous-positive strain, reactions were abolished. It appears that reactions of anti-E. coli J5 antiserum and anti-S. typhimurium Ra antiserum with homologous and heterologous strains were not due to the same antibody. This immunoblotting technique proved to be a useful method to distinguish different antibodies in antiserum raised against LPS of gram-negative bacteria.