Rationale for a Swedish cohort consortium

We herein outline the rationale for a Swedish cohort consortium, aiming to facilitate greater use of Swedish cohorts for world-class research. Coordination of all Swedish prospective population-based cohorts in a common infrastructure would enable more precise research findings and facilitate research on rare exposures and outcomes, leading to better utilization of study participants' data, better return of funders' investments, and higher benefit to patients and populations. We motivate the proposed infrastructure partly by lessons learned from a pilot study encompassing data from 21 cohorts. We envisage a standing Swedish cohort consortium that would drive development of epidemiological research methods and strengthen the Swedish as well as international epidemiological competence, community, and competitiveness.Peer reviewe

Differences in Genetic and Transcriptional Organization of the glpTQ Operons between Haemophilus influenzae Type b and Nontypeable Strains

The glpTQ operon of Haemophilus influenzae type b (Hib) and nontypeable H. influenzae (NTHi) strains is highly conserved, except for a 1.4-kb glpTQ intergenic region that was found in most Hib strains. The presence of this intergenic region results in divergent glpTQ transcriptional profiles for Hib and NTHi where Hib strains appear to have evolved an alternative promoter for glpQ expression. Based on the intergenic region's low G+C content, we speculate that this DNA fragment was acquired by lateral transfer

Protein D of Haemophilus influenzae: a protective nontypeable H. influenzae antigen and a carrier for pneumococcal conjugate vaccines.

Protein D (PD) is a highly conserved 42 kDa surface lipoprotein found in all Haemophilus influenzae, including nontypeable (NT) H. influenzae. PD is involved in the pathogenesis of respiratory tract infections, in the context of which it has been shown to impair ciliary function in a human nasopharyngeal tissue culture model and to augment the capacity to cause otitis media in rats. A likely mechanism indicating that PD is a virulence factor is its glycerophosphodiesterase activity, which leads to the release of phosphorylcholine from host epithelial cells. PD has been demonstrated to be a promising vaccine candidate against experimental NT H. influenzae infection. Rats vaccinated with PD cleared NT H. influenzae better after middle ear and pulmonary bacterial challenge, and chinchillas vaccinated with PD showed significant protection against NT H. influenzae-dependent acute otitis media. In a clinical trial involving children, PD was used as an antigenically active carrier protein in an 11-valent pneumococcal conjugate investigational vaccine; significant protection was achieved against acute otitis media not only caused by pneumococci but also caused by NT H. influenzae. This may have great clinical implications, because PD is the first NT H. influenzae antigen that has induced protective responses in humans

Glycerol-3-phosphate transport in Haemophilus influenzae: cloning, sequencing, and transcription analysis of the glpT gene

The presence of a functional glpT gene in Haemophilus influenzae could be questioned, since there is only what appears to be a truncated glpT (HI0686, 143 nt in the 5'-end) available in the H. influenzae Rd genome database (Fleischmann et al. , 1995). For cloning of the glpT gene from H. influenzae type b strain Eagan, an isogenic glpT, rec-1 double mutant and a selective medium for detection of the glpT mutant strains were constructed. The recombinant plasmid carrying glpT was able to complement the isogenic glpT mutant to wild-type levels of G3P uptake and permitted growth on a selective medium with G3P as a major carbon source. The nucleotide sequences of the glpT gene were determined both directly from PCR products and from the cloned DNA insert of strain Eagan. An identical 1440 bp open reading frame with 480 deduced amino acids, highly homologous to other bacterial G3P permeases, was identified. A Northern blot analysis showed that the glpT genes in both Eagan and Rd strains were transcribed on a RNA of approximately 1.4 kb in size. Thus, it is likely that HI0686 sequence originates from a mutated glpT clone in Escherichia coli

Fragmentation heterogeneity of 23S ribosomal RNA in Haemophilus species

The fragmentation of 23S rRNA of 23 Haemophilus influenzae strains and eight strains belonging to other Haemophilus species was investigated. Instead of intact molecules, the 23S rRNA molecules were found to be cleaved into two to five smaller conserved fragments in most strains examined, especially in H. influenzae type b (5/6) and nontypeable strains (5/5). One or two conserved potential cleavage sites were identified by PCR analysis of the strains showing a fragmented 23S rRNA pattern. The relevant nucleotide sequences were determined and compared to H. influenzae Rd, which contains intact 23S rRNA molecules. An identical 112bp long intervening sequence (IVS) at position 542 and a conserved 121-123bp IVS sequence at position 1171 were found in two H. influenzae type b strains and one nontypeable strain. Among the strains with fragmented 23S rRNA, nearly half showed a heterogeneous cleavage pattern due to the dispersion of IVSs among different 23S rRNA operons. The localization of the conserved H. influenzae IVSs coincided well with the extensively studied IVSs among other bacteria, but differed in nucleotide sequence from any other reported IVSs. Therefore, the IVSs of Haemophilus 23S rRNA may originate from a common source that is independent of other bacteria

Biological activity of serum antibodies to a nonacylated form of lipoprotein D of Haemophilus influenzae

Protein D, a surface-exposed 42-kDa membrane lipoprotein, is well conserved among both type b and nontypeable Haemophilus influenzae strains, and it is considered a vaccine against H. influenzae infections. Here, we report the large-scale purification of a nonacylated form of protein D (PDm) from the periplasmic space of Escherichia coli overexpressing PDm. Screening of human sera for levels of antibodies to PDm demonstrated that the immunoglobulin G (IgG) antibody level is above background levels in infants less than 6 months of age. Following a drop to background values in the age group 6 months to 1 year, IgG antibody levels start to increase, together with IgA antibody levels, after 1 year of age. The first appearance of serum IgM antibodies is in 6-month- to 1-year-old infants whose IgG antibody levels have dropped to the postnatal background level. Affinity-purified antibodies from humans and from PDm-immunized rats detected epitopes of protein D which are normally exposed on the bacterial surface. Affinity-isolated human anti-PDm antibodies eluted in acidic buffer were not bactericidal against H. influenzae. Loss of bactericidal activity may occur in this buffer, as was demonstrated in pooled human sera with high bactericidal activity after incubation in the same buffer. Hyperimmunization of rats with PDm induced high levels of serum IgG and IgA antibodies against PDm and significant bactericidal activity against homologous and heterologous H. influenzae strains

Debridement of Bacterial Biofilms with TiO2/H2O2 Solutions and Visible Light Irradiation

Objectives. The aim of the study was to explore the debridement efficacy of different solutions of H2O2 and rutile particles against Staphylococcus epidermidis and Pseudomonas aeruginosa biofilms attached to titanium surfaces when exposed to visible light. Materials and Methods. Titanium discs cultivated with biofilms of Staphylococcus epidermidis or Pseudomonas aeruginosa were subjected for 1 min to suspensions consisting of rutile particles mixed with high (950 mM) or low (2 mM) concentrations of H2O2 under visible light irradiation (405 nm; 2.1 mW/cm2). Discs were rinsed and the degree of debridement was determined through scanning electron microscopy and viability assessment of the remaining bacteria using luminescence measurements and/or a metabolic activity assay. Results. Cleaning mixtures containing the higher concentration of H2O2 showed a significantly improved debridement compared to the negative control in all experiments. The addition of rutile particles was shown to have a statistically significant effect in one test with S. epidermidis. Limited evidence of the catalytic effect of visible light irradiation was seen, but effects were relatively small and statistically insignificant. Conclusions. H2O2 at a concentration of 950 mM proved to be the strongest contribution to the debridement and bactericidal effect of the cleaning techniques tested in this study

Protein D, the glycerophosphodiester phosphodiesterase from Haemophilus influenzae with affinity for human immunoglobulin D, influences virulence in a rat otitis model

A mutant lacking the ability to express the surface-exposed lipoprotein protein D was constructed by linker insertion and deletion mutagenesis of a cloned DNA insert containing the protein D structural gene from a nontypeable Haemophilus influenzae strain (NTHi). An isogenic NTHi mutant was isolated after transformation of genetically competent bacteria. The transformant was unreactive to a protein D-specific monoclonal antibody in a colony immunoassay. In addition, the mutant lacked the ability to synthesize detectable levels of protein D by protein staining, immunoblot methods, glycerophosphodiester phosphodiesterase activity, and binding studies of radiolabelled immunoglobulin D. The isogenic protein D-deficient mutant was compared with its parental strain for its ability to induce experimental otitis media in rats challenged with bacteria. An approximately 100-times-higher concentration of the mutant compared with that of the wild-type strain was required in order to cause otitis among all rats challenged with that given dose. The protein D mutant exhibited a generation time that was equal to that of the wild-type strain in complex broth medium. No difference in lipopolysaccharide expression was found between the mutant and the parental strain. These results suggest that protein D may influence the pathogenesis of NTHi in the upper respiratory tract

Protein D expression promotes the adherence and internalization of non-typeable Haemophilus influenzae into human monocytic cells

Protein D, having a glycerol-3-phosphodiester phosphodiesterase activity, is found at the surface of all Haemophilus influenzae strains and is a possible virulence factor. In the present study, the involvement of protein D in the entry of NTHi into human monocytic cells is reported. Primary monocytes and the monocytic cell lines U-937 and THP-1 were infected with NTHi strain 772 and the mutant 772 Delta hpd 1 (lacking the gene for protein D). NTHi 772 adhered to and entered monocytic cells up to four-fold more efficiently compared to 772 Delta hpd 1. When an Escherichia coli transformant expressing protein D was incubated with monocytic cells, the number of intracellular bacteria increased 1.6-fold compared to protein D-deficient controls. Any correlation between internalization and phosphorylcholine expression was not detected. In conclusion, our data suggest that surface-expressed protein D promotes the adherence of NTHi to human monocytes leading to a higher number of internalized bacteria