To gain insight into the defects responsible for impaired Th1 responses in human newborns, we analyzed the production of cytokines by dendritic cells (DC) derived from cord blood monocytes. We observed that neonatal DC generated from adherent cord blood mononuclear cells cultured for 6 days in the presence of IL-4 and GM-CSF show a phenotype similar to adult DC generated from adherent PBMC, although they express lower levels of HLA-DR, CD80, and CD40. Measurement of cytokine levels produced by neonatal DC upon stimulation by LPS, CD40 ligation, or poly(I:C) indicated a selective defect in the synthesis of IL-12. Determination of IL-12(p40) and IL-12(p35) mRNA levels by real-time RT-PCR revealed that IL-12(p35) gene expression is highly repressed in stimulated neonatal DC whereas their IL-12(p40) gene expression is not altered. The addition of rIFN-γ to LPS-stimulated newborn DC restored their expression of IL-12(p35) and their synthesis of IL-12 (p70) up to adult levels. Moreover, we observed that neonatal DC are less efficient than adult DC to induce IFN-γ production by allogenic adult CD4+ T cells. This defect was corrected by the addition of rIL-12. We conclude that neonatal DC are characterized by a severe defect in IL-12(p35) gene expression which is responsible for an impaired ability to elicit IFN-γ production by T cells.
The induction of dendritic cell (DC) maturation is critical for the induction of Ag-specific T lymphocyte responses and may be essential for the development of human vaccines relying on T cell immunity. In this study, we have investigated the effects of monophosphoryl lipid A (MPL) on human monocyte-derived DC as well as peripheral blood T cells. Calcium mobilization, mitogen-activated protein kinase activation, and the NF-κB transcription factor were induced after MPL stimulation of DC and required high doses of MPL (100 μg/ml). Maturation parameters such as production of IL-12 and increases in cell surface expression of HLA-DR, CD80, CD86, CD40, and CD83 were observed following DC treatment with MPL. However, lower levels of IL-12 were induced by MPL when compared with lipopolysaccharide. This is likely to be related to differences in the kinetics of extracellular signal-related kinase 1/2 and p-38 phosphorylation induced by both molecules. Although maturation induced by MPL was weaker when compared with lipopolysaccharide, it appeared to be sufficient to support optimal activation of allogeneic naive CD45RA+ T cell and anti-tetanus toxoid CD4 T cells. MPL at low doses (5 μg/ml) had no impact on DC maturation, while its addition to DC-T cell cocultures induced full T cell activation. The observed effect was related to the fact that MPL also acts directly on T cells, likely through their Toll-like receptors, by increasing their intracellular calcium and up-regulating their CD40 ligand expression. Together, these data support a model where MPL enhances T cell responses by having an impact on DC and T cells.
Although the PCR for the detection of Bordetella pertussis is routinely performed in diagnostic laboratories, no quality assessment program has so far been described. We report on the results obtained with two external quality assessment proficiency panels sent to European laboratories. The first proficiency panel contained a series of dilutions of three previously characterized B. pertussis clinical isolates and two negative controls. No false-positive results were reported by six laboratories providing seven data sets. The reported limits of detection of the three B. pertussis strains varied between 4 and 4,000, 9 and 9,000, and 3 and 30,000 CFU/ml, respectively. The second proficiency panel, composed of a series of dilutions of reference strains of B. pertussis, B. holmesii, B. hinzii, and B. bronchiseptica, as well as negative controls, was sent to nine laboratories. One laboratory reported a negative result for a sample and reported a B. parapertussis-positive sample to be positive for B. pertussis. By using the B. pertussis-specific target gene pertactin, one laboratory detected B. pertussis with 100% specificity. All other laboratories, which used IS481-based assays, reported positive results for the samples containing B. holmesii and B. bronchiseptica, species that have occasionally been recovered from human respiratory samples. These data show that the choice of the target gene is particularly critical for the species specificity of B. pertussis PCR assays.
A novel real-time PCR (RT-PCR) assay was developed for detection of Bordetella pertussis in respiratory specimens by targeting the pertactin gene. In vitro evaluation with reference strains and quality control samples showed analytical sensitivity equivalent to and specificity superior to those of PCR assays which target the IS481 element. The pertactin-based RT-PCR assay offers better discrimination between B. pertussis and other Bordetella species than previously described assays. INTRODUCTIONBordetella pertussis is the aetiological agent of whooping cough or pertussis. In the non-vaccinated population, it primarily affects children less than 6 months of age. The most common manifestations of B. pertussis infection are whooping cough and bronchitis with complications including pneumonia, seizures, encephalopathy and possibly sudden infant death (Mattoo & Cherry, 2005). During the last 15 years, the incidence of pertussis has increased again in different regions of the world despite intensive programmes of infant vaccination (Cordova et al., 2000;De Melker et al., 2000;De Schutter et al., 2003;Gonzalez Moran et al., 2002;Khetsuriani et al., 2001). Adults and adolescents act as a reservoir for infection in very young infants who are not yet fully immunized and who experience severe morbidity. This reservoir exists because vaccination in childhood induces protection for a limited time only (Mattoo & Cherry, 2005;Von Konig et al., 2002). The emergence and dissemination of B. pertussis variant strains that are antigenically different from vaccine strains is another possible cause of this phenomenon (Weber et al., 2001).In this context, rapid and accurate diagnostic tests for pertussis appear to be needed for improved management of cases and protection of infants. Most of the PCR-based methods described so far for the detection of B. pertussis target the multi-copy insertion sequence IS481 (Chan et al., 2002; Cloud et al., 2003;Poddar, 2003;Reischl et al., 2001), which is also found in other Bordetella species (Muyldermans et al., 2005). Here, we describe a novel PCR assay for specific detection of B. pertussis that we have designed by targeting the single-copy pertactin gene, a 69 kDa outer-membrane protein which is an important virulence factor of B. pertussis. The real-time pertactin PCR (pertactin RT-PCR) can be combined with sequence analysis of 16S rRNA genes for the identification of other species of Bordetella. METHODSBacterial isolates. The assay specificity was evaluated on strains of Bordetella (n58) and non-Bordetella species (n520), including: Bordetella pertussis (ATCC 9340 and 9797), Bordetella parapertussis, Bordetella bronchiseptica, Bordetella holmesii, Bordetella hinzii, Bordetella trematum, Bordetella avium, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Streptococcus pneumoniae, Stenotrophomonas maltophilia, Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Morganella morganii, Enterobacter aerogenes, Citrobacter freundii, Proteus vulgaris, Burkh...
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