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Lyme borreliosis is characterized by cellular inflammatory responses at multiple body sites. Recently, an association of interleukin-17 (IL-17) and Lyme arthritis was suggested. In this context, it is of special interest that the heterodimeric cytokine IL-23 can act on T cells and initiate the up-regulation of effector cytokines such as IL-17. To determine the role of this specific cytokine cascade for the induction of subsequently induced proinflammatory events we developed an in vitro system to investigate the IL-23-inducing capacity of Borrelia burgdorferi and the potential of the spirochete for inducing the IL-23/IL-17 axis. We used cells derived from mice deficient for IL-23 or IL-12 only or deficient for both IL-12 and IL-23 to define precisely the function of these cytokines. Experiments with bone marrow-derived dendritic cells (BMDC) identified these cells as sources for IL-23 but not for IL-12 after B. burgdorferi exposure. Subsequent investigations with T cell-depleted splenocyte fractions revealed a tight IL-23/IL-17 axis in response to the spirochetes. Monoclonal antibodies that block IL-23 showed further that BMDC-derived IL-23 was required for production of IL-17 in this experimental model. These in vitro data describing a spirochete-induced release of IL-23 may help to define IL-17-dependent inflammatory responses in the disease.

The receptor for advanced glycation end-products (RAGE) and its soluble forms are predominantly expressed in lung but its physiological importance in this organ is not yet fully understood. Since RAGE acts as a cell adhesion molecule, we postulated its physiological importance in the respiratory mechanics. Respiratory function in a buffer-perfused isolated lung system and biochemical parameters of the lung were studied in young, adult, and old RAGE knockout (RAGE-KO) mice and wild-type (WT) mice. Lungs from RAGE-KO mice showed a significant increase in the dynamic lung compliance and a decrease in the maximal expiratory air flow independent of age-related changes. We also determined lower mRNA and protein levels of elastin in lung tissue of RAGE-KO mice. RAGE deficiency did not influence the collagen protein level, lung capillary permeability, and inflammatory parameters (TNF-␣, high-mobility group box protein 1) in lung. Overexpressing RAGE as well as soluble RAGE in lung fibroblasts or cocultured lung epithelial cells increased the mRNA expression of elastin. Moreover, immunoprecipitation studies indicated a trans interaction of RAGE in lung epithelial cells. Our findings suggest the physiological importance of RAGE and its soluble forms in supporting the respiratory mechanics in which RAGE trans interactions and the influence on elastin expression might play an important role.receptor for advanced glycation end-products; aging; biomechanics; elastin; mouse THE RECEPTOR FOR ADVANCED glycation end-products (RAGE) is a pattern recognition receptor of the immunoglobulin superfamily (32). RAGE is predominantly expressed in lung, particularly in the type I alveolar epithelial (ATI) cells (12,15,46). This specific localization suggests its important physiological function in the alveolar epithelium. In other cell types and tissues, the expression of RAGE is activated in response to pathophysiological conditions, such as the accumulation of advanced glycation end-products (AGEs) and inflammation (1). RAGE expression is highly associated with lung development and increases during the alveolarization (29,40). High RAGE expression prior to the alveolarization period has adverse effects associated with a severe pulmonary dysplasia (16,41). A reduced alveolar expression of RAGE is related to pathophysiological changes of the lung tissue, such as carcinoma (3), fibrosis (34, 37), and chronic obstructive pulmonary disease (COPD) (34). In addition to the membrane-bound receptor, soluble RAGE (sRAGE) forms exist as the result of either alternative splicing (22) or protein shedding by metalloproteinases (39, 52). sRAGE normally exists in the bronchoalveolar lavage (BAL) at a high level (53), but it is deficient in neutrophilic asthma and COPD (47, 49).The physiological importance of RAGE in lung is not yet fully understood because mice lacking RAGE do not show obvious pulmonary alterations (5, 9). Only intervention studies indicated an adverse effect of RAGE in the development of lung fibrosis (14, 19) and acute lung injury (4...

BackgroundStudies on osteoclasts, the bone resorbing cells, have remained limited due to the lack of transgenic mice allowing the conditional knockout of genes in osteoclasts at any time during development or adulthood.Methodology/Principal FindingWe report here on the generation of transgenic mice which specifically express a tamoxifen-inducible Cre recombinase in osteoclasts. These mice, generated on C57BL/6 and FVB background, express a fusion Cre recombinase-ERT2 protein whose expression is driven by the promoter of cathepsin K (CtsK), a gene highly expressed in osteoclasts. We tested the cellular specificity of Cre activity in CtsKCreERT2 strains by breeding with Rosa26LacZ reporter mice. PCR and histological analyses of the CtsKCreERT2LacZ positive adult mice and E17.5 embryos show that Cre activity is restricted largely to bone tissue. In vitro, primary osteoclasts derived from the bone marrow of CtsKCreERT2+/−LacZ+/− adult mice show a Cre-dependent β-galactosidase activity after tamoxifen stimulation.Conclusions/SignificanceWe have generated transgenic lines that enable the tamoxifen-induced, conditional deletion of loxP-flanked genes in osteoclasts, thus circumventing embryonic and postnatal gene lethality and avoiding gene deletion in other cell types. Such CtsKCreERT2 mice provide a convenient tool to study in vivo the different facets of osteoclast function in bone physiology during different developmental stages and adulthood of mice.

The 1282 bp cDNA of an isoenzyme of fructose-1,6-bisphosphatase was cloned from rat muscle. It shows 70% positional identity to the cDNA of rat liver fructose-1,6-bisphosphatase and is clearly the product of a gene different from that coding for the liver enzyme. After cloning of the coding region of the rat muscle fructose-1,6-bisphosphatase cDNA in an expression vector, the recombinant enzyme could be detected in E. coli cell-free extracts by activity determination and Western blotting. Overexpressed fructose-1,6-bisphosphatase was found to be allosterically inhibited by AMP comparably to the enzyme isolated from rat muscle. Analysis of steady-state mRNA levels of various rat tissues with reverse-transcriptase polymerase chain reaction (RT-PCR) and Northern blotting revealed one or the two fructose-1,6-bisphosphatase isoenzyme mRNAs in most tissues tested with significant quantitative differences. Quantitative PCR using a homologous competitor showed that 1 microg of total RNA of rat muscle contains 1.7 x 10(6) molecules of rat muscle fructose-1,6-bisphosphatase mRNA. 3 x 10(4) copies of this message were found per microg total RNA of heart and kidney, respectively.

A rapid competitive PCR method was developed to quantify DNA on the LightCycler. It rests on the quantitative information contained in the melting curves obtained after amplification in the presence of SYBR Green I. Specific hybridization probes are not required. Heterologous internal standards sharing the same primer binding sites and having different melting temperatures to the natural PCR products were used as competitors. After a co-amplification of known amounts of the competitor with a DNA-containing sample, the target DNA can be quantified from the ratio of the melting peak areas of competitor and target products. The method was developed using 16S rDNA fragments from Streptococcus mutans and E. coli and tested against existing PCR-based DNA quantification procedures. While kinetic analysis of real-time PCR is well established for the quantification of pure nucleic acids, competitive PCR on the LightCycler based on an internal standardization was found to represent a rapid and sensitive alternative DNA quantification method for analysis of complex biological samples that may contain PCR inhibitors.

Borrelia burgdorferi, the agent of Lyme borreliosis, has the ability to undergo morphological transformation from a motile spirochetal to non-motile spherical shape when it encounters unfavorable conditions. However, little information is available on the mechanism that enables the bacterium to change its shape and whether major components of the cells--nucleic acids, proteins, lipids--are possibly modified during the process. Deducing from investigations utilizing electron microscopy, it seems that shape alteration begins with membrane budding followed by folding of the protoplasmatic cylinder inside the outer surface membrane. Scanning electron microscopy confirmed that a deficiency in producing functioning periplasmic flagella did not hinder sphere formation. Further, it was shown that the spirochetes' and spheres' lipid compositions were indistinguishable. Neither phosphatidylcholine nor phosphatidylglycerol were altered by the structural transformation. In addition, no changes in differential protein expression were detected during this process. However, minimal degradation of RNA and a reduced antigen-antibody binding activity were observed with advanced age of the spheres. The results of our comparisons and the failure to generate mutants lacking the ability to convert to spheres suggest that the metamorphosis of B. burgdorferi results in a conditional reconstruction of the outer membrane. The spheres, which appear to be more resistant to unfavorable conditions and exhibit reduced immune reactivity when compared to spirochetes, might allow the B. burgdorferi to escape complete clearance and possibly ensure long-term survival in the host.