Conditional transgenic mouse models: from the basics to genome-wide sets of knockouts and current studies of tissue regeneration.

Many mouse models are currently available, providing avenues to elucidate gene function and to recapitulate specific pathological conditions. To a large extent, successful translation of clinical evidence or analytical data into appropriate mouse models is possible through progress in transgenic or gene-targeting technology. Beginning with a review of standard mouse transgenics and conventional gene targeting, this article will move on to discussing the basics of conditional gene expression: the tetracycline (tet)-off and tet-on systems based on the transactivators tet-controlled transactivator (Tta) and reverse tet-on transactivator (rtTA) that allow downregulation or induction of gene expression; Cre or Flp recombinase-mediated modifications, including excision, inversion, insertion and interchromosomal translocation; combination of the tet and Cre systems, permitting inducible knockout, reporter gene activation or activation of point mutations; the avian retroviral system based on delivery of rtTA specifically into cells expressing the avian retroviral receptor, which enables cell type-specific, inducible gene expression; the tamoxifen system, one of the most frequently applied steroid receptor-based systems, allows rapid activation of a fusion protein between the gene of interest and a mutant domain of the estrogen receptor, whereby activation does not depend on transcription; and techniques for cell type-specific ablation. The diphtheria toxin receptor system offers the advantage that it can be combined with the 'zoo' of Cre recombinase driver mice. Having described the basics we move on to the cutting edge: generation of genome-wide sets of conditional knockout mice. To this end, large ongoing projects apply two strategies: gene trapping based on random integration of trapping vectors into introns leading to truncation of the transcript, and gene targeting, representing the directed approach using homologous recombination. It can be expected that in the near future genome-wide sets of such mice will be available. Finally, the possibilities of conditional expression systems for investigating gene function in tissue regeneration will be illustrated by examples for neurodegenerative disease, liver regeneration and wound healing of the skin.

Neisseria meningitidis, Neisseria lactamica and Moraxella catarrhalis share cross-reactive carbohydrate antigens.

Carriage of commensal bacteria species is associated with the development of natural immunity to meningococcal disease, with lipo-oligosaccharides (LOS) of meningococci being one of the main virulence factors associated with severity of meningococcal disease. Meningococcal reference strains and isolates from the commensal species Neisseria lactamica and Moraxella catarrhalis were assessed for the presence of cross-reactive glycoconjugate antigens. Binding of human blood group antibodies of the P and Ii system to meningococcal immunotype reference strains were in accordance with the presence of known LOS carbohydrate structures. Binding studies with meningococcal immunotyping antibodies and blood group phenotyping antibodies to N. lactamica strains from different European countries showed, that a greater number of isolates obtained from native Greek and Scottish adults and children bound anti-meningococcal L(3, 7, 9) immunotyping (P < 0.001), pK (P = 0.035) and paragloboside (P < 0.001) blood group typing antibodies compared to isolates obtained from children of Russian immigrants in Greece. A greater number of M. catarrhalis strains isolated from children in Scotland bound anti-L(3, 7, 9) antibodies (38.2%) compared to strains isolated from adults (22.2%) (P = 0.017). These findings provide evidence that blood group like glycoconjugate antigens found on the commensal species N. lactamica and M. catarrhalis might be involved in the development of natural immunity to meningococcal endotoxins during childhood, and might be exploited as anti-meningococcal vaccine candidates.

Proinflammatory responses to lipo-oligosaccharide of Neisseria meningitidis immunotype strains in relation to virulence and disease.

Inflammatory responses to lipo-oligosaccharide (LOS) contribute to the severity of meningococcal disease. Strains that express the L(3,7,9) LOS immunotypes are isolated from the majority of patients, but other immunotypes are isolated predominantly from carriers. Inflammatory responses elicited from a human monocytic cell line (THP-1) that had been pretreated with vitamin D3 (VD3) were compared after stimulation with purified LOSs from standard immunotype strains. The neutralizing effects of normal human serum and serum from mice immunized with strain B:2a:P1.5,2:L3 were compared. LOSs of immunotypes L3, L7, L8, and L9 induced significantly higher levels of tumor necrosis factor-alpha and interleukin-6, compared with other immunotypes. Normal human serum neutralized the proinflammatory responses to LOSs of all immunotypes tested. Immune mouse serum neutralized inflammatory responses against LOSs from immunotypes with epitopes cross-reactive with L(3,7,9) moieties. Antibodies found in normal human serum and immune mouse serum to the oligosaccharide, core, and lipid A moieties of meningococcal endotoxin contribute to neutralizing activity.

Blood group and susceptibility to disease caused by Escherichia coli O157.

Patients (n=186) infected during the Escherichia coli O157 outbreak in Scotland in 1996 were assessed for blood group markers (ABO, Lewis, and P) associated with other gastrointestinal infections. Binding of bacteria to epithelial cells was assessed by flow cytometry. Buffy coats from blood donors were examined for inflammatory responses to culture filtrates of the outbreak strain. Individuals of blood group O comprised 63.4% of patients, compared with 53.4% (P <.05) and 53.9% (P <.01) of neighboring populations in Airdrie and Glasgow, respectively; group O also comprised 64.3% of patients with hemolytic uremic syndrome (HUS) and 87.5% of patients who died (P <.05). No or weak agglutination by anti-P antiserum was observed for 40.7% of control persons (n=122), 61.5% of all patients (P =.0027), and 83.3% of patients with HUS (P =.013). The susceptibility of group O to E. coli was not associated with increased binding of bacteria to epithelial cells or with higher production of tumor necrosis factor (TNF)-alpha or interleukin-6. Leukocytes of P-negative blood donors produced higher levels of TNF-alpha than those of P-positive donors.