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The energetic state of Ips typographus L. (Col., Scolytidae) during the life cycle. The energetic state of adults of the European spruce engraver I. typographus was investigated during different stages of the life cycle: maturation feeding, hibernation, dispersal, and just after starting to excavate brood galleries. The beetles were characterized by the pattern of free amino acids and especially by the concentrations of proline and alanine. Additionally the lipid concentrations were estimated. In comparison to all adults investigated, only newly moulted beetles showed the highest content of amino acids and a large variety of those amino acids which can be integrated in proteins. The process of sclerotization during the maturation feeding was accompanied by an increase in lipid content and a decrease in the amount of amino acids, with the exception of proline and alanine. In all further physiological stages of the beetles, proline and alanine were found to be dominant. Proline and alanine constituted, in total, 70-80 % of the free amino acids. During dispersal, the sum of proline and alanine ranged between 95 and 130 nmol/mg dry matter, whereas, before and after hibernation, as well as in breeding adults, the corresponding values varied between 60 and 75 nmol/mg dry matter. The concentrations of alanine reached considerably higher amounts in the flying stages. Depending on the date of emergence, the lipid content of the adults (expressed as μequi × acylesters/mg dry matter) varied as follows: 0.32, 0.75 and 0.89. During hibernation, the lipid content was reduced by about 60 %. The results indicated that flying adults have a proline/alanine shuttle system by which energy-rich compounds derived from the degradation of lipids are distributed within the insect. In line with this hypothesis, the energetic state of flying beetles is characterized by two parameters: 1. The lipid content representing the total energy reserves which are potentially available; and 2. The ratio of the concentrations of proline and alanine being regarded as a measure of the energy which is actually available for respiration during flight. Increasing values of the proline/alanine-ratio indicated higher levels of fuels for respiration. This approach was used to characterize the energetic state of dispersing beetles. During dispersal, attacked trap-trees seemed to be more attractive to I. typographus, especially to females, than pheromone traps. Zusammenfassung Der energetische Status von I. typographus wurde während der Entwicklung der Jungkäfer unter der Rinde sowie in weiteren populationsdynamisch relevanten Lebensphasen (Dispersion, Eiablage, überwinterung) untersucht. Hierzu wurden die freien Aminosäuren, insbesondere Prolin und Alanin sowie die Acylester als Maß für die Fettreserven bestimmt. Noch nicht ausgefärbte Jungkäfer besaßen im Gegensatz zu allen anderen untersuchten physiologischen Stadien relativ hohe Gehalte an freien Aminosäuren. Nach der Ausfärbung der Jungkäfer dominierten Prolin und Alanin mit einem gemeinsamen molaren Anteil von 70-80%. In der Dispersionsphasce betrugen ihre Absolutgehalte in der Summe 95-130 nmol/mg TM. Zu Beginn der Brut sowie vor und nach der überwinterung lagen die entsprcchenden Werte bei durch-schnittlich 60-75 nmol mg TM. Auffällig war vor allem bei grundsätzlich flugbereiten Käferstadien der vergleichsweise hohe Alaningehalt. Während der Ausfärbung reicherten die Imagines Lipide an. Ausgefärbte Jungkäfer, die die Brutsysteme verließen, besaßn in Abhängigkeit vom Ausbohrtermin die folgenden durchschnittlichen Lipidgehalte in μmol Acylester mg TM: 0,32 (August). 0,75 (September). 0,89 (Oktober). Letztgenannter Wert der insgesamt höchsle der gefundenen entsprach 26,5 Gewichtsprozent. Durch die überwinterung verminderten sich die Lipidvorräte um ca. 60 %. Die Ergebnisse liefern Hinweise, daß die in den Lipiden gespeicherte Energie über einen Prolin/Alanin-shuttle für die Atmung bereitgestellt wird. Auf dieser Basis wird der energetische Status dispergierender Käfer durch 2 Parameter charakterisiert: 1. Das insgesamt verfügbare Energiepotential durch die Gehalte an Acylestern; und 2. Die aktuell für die Atmung verfügbare Energie durch den Quotienten aus den Konzentrationen von Prolin und Alanin (Pro/Ala). Hohe Werte für Pro/Ala indizieren ein hohes Maß an aktuell verfügbarer Energie. Mit Hilfe dieser Meßgrößen wurde der energetische Zustand des Buchdruckers charakterisiert. Für die Dispersions- und Besiedelungsphase zeichnete sich die Tendenz ab, daß frisch bebrütete Fangbäume-insbesondere für Weibchen-stärker dispersionsverkürzend wirken als Pheromonfallen. [ABSTRACT FROM AUTHOR]

Interferon-gamma (IFNgamma)-mediated indoleamine 2,3-dioxygenase (IDO) expression, important in innate immunity, immune suppression, and tolerance, can be counteracted by ferrous iron (FeSO(4)). Elevation of intracellular iron levels during stimulation with IFNgamma impeded IFNgamma-induced IDO mRNA and protein expression in HEp-2 cells. Decreased IDO expression was accompanied by decreased tryptophan degradation. Accordingly, IFNgamma-mediated suppressing effects on Chlamydia trachomatis (CT) infection were reduced or even abolished in the presence of FeSO(4). Conversely, lowering intracellular iron levels by deferoxamine (DFO) did not increase IFNgamma-induced IDO expression but potentiated Chlamydia-suppressing effects by lowering intracellular iron availability. Additionally, DFO led to a CT-induced IDO expression in HEp-2 cells not treated with IFNgamma. In summary, this study demonstrates that iron acts as a regulatory element for modulating IDO expression, in addition to its function as an essential element for chlamydial growth. This may represent an important control mechanism of IDO expression at the transcriptional level.

Chlamydophila pneumoniae (C. pneumoniae) has been detected in macrophages (Mø) and dendritic cells (DC) in vascular diseases. To understand the importance of these cell types in C. pneumoniae infection and transmission, we infected DC and cultivated them with Mø in a coculture model system which precludes cell-to-cell contact during chlamydial infection. C. pneumoniae inside living DC were labeled and tracked with a red fluorescent ceramide dye. Subsequently, red-coloured chlamydial inclusions were detected 3 and 5 days later in cocultured Mø. Moreover, standard assays revealed infectious elementary bodies in infected DC and cocultured Mø. Assays for chlamydial gene expression indicated vital and dividing chlamydiae in both cell types. In summary, the results suggest that the transwell system employed here is a suitable model to investigate the transmission of C. pneumoniae from DC to Mø. Importantly, the observations presented demonstrate that transmission is independent of cell-to-cell contact.

Peptidomic analysis using Differential Peptide Display (DPD) of human peripheral blood mononuclear cells (PBMC) mock-infected or persistently infected by Chlamydia trachomatis (CT) revealed 10 peptides, expressed upon CT infection. Analysis of these 10 candidates by tandem mass spectrometry enabled the determination of seven candidates as fragments from the precursors (I) ferritin heavy chain subunit, (II) HLA class II histocompatibility antigen, (III) vimentin, (IV) indoleamine 2,3-dioxygenase, (V and VI) pre-B cell enhancing factor (PBEF), and (VII) Interleukin-8 (CXCL8). The identified candidates proved the presence of anti-bacterial and immunologically active monocytic proteins after CT infection.

Earlier studies from this group demonstrated that Chlamydophila pneumoniae co-localized with dendritic cells (DC) in temporal artery biopsies from patients with giant cell arteritis (GCA). To investigate the interaction of DC with C. pneumoniae we employed an in vitro cell culture system of human monocyte derived DC. These DC were infected with C. pneumoniae and observed at regular time intervals up to 25 days post infection. Chlamydiae were visualized inside DC by both confocal and electron microscopy. Statistical analysis showed an increase in the number of chlamydial antigen during that period (p < 0.00005, chi2-test). Titration of DC lysates on HEp-2 cells showed that infectious progeny was recovered at various intervals but showed no exponential growth. Additionally, RT-PCR analyses of infected DC identified transcripts from dnaA, ftsK and tal throughout a period of 14 days, indicating viable chlamydiae. Thus, human monocyte-derived DC are susceptible to C. pneumoniae infection. These results indicate that C. pneumoniae-infected DC can play an important role in the transmission of these bacteria in GCA and other chlamydial diseases.

Chlamydia trachomatis (CT) as well as Chlamydophila pneumoniae (CP) cause chronic inflammatory diseases in humans. Persistently infected monocytes are involved in the pathogenesis by inducing mediators of inflammation. An in vitro system of chlamydial persistence in human peripheral blood monocytes (HPBM) was used to investigate prostaglandin E(2) (PGE(2)) production and the expression of the key enzyme for prostaglandin production, cyclooxygenase-2 (COX-2). PGE(2) production was determined by PGE(2)-ELISA of HPBM-culture supernatants. Cox-2 mRNA expression was measured by real-time RT-PCR of total RNA isolated from HPBM. Both, CT and CP, stimulated PGE(2) production of HPBM in vitro. Equivalent numbers of CT per host cell induced a higher PGE(2)-response compared to CP. The amount of synthesized PGE(2) depended on the chlamydial multiplicity of infection (MOI). Even at an MOI of 10 the amount of CT- and CP-induced prostaglandin, respectively, was lower than the amount of prostaglandin induced by E. coli lipopolysaccharide (LPS) at a concentration of 10microg/ml. In contrast to stimulation with LPS, Chlamydia-induced PGE(2) production as well as cox-2 mRNA decreased after day 1 post infection (p.i.). These data indicate that Chlamydia stimulate PGE(2) production in human monocytes. Since Chlamydia are often contaminated by mycoplasma, the influence of mycoplasma on the prostaglandin production was investigated additionally. Mycoplasma fermentans (MF) also stimulated PGE(2) production. The co-infection of mycoplasma and Chlamydia resulted in an additive effect in the production of PGE(2). Thus it is important to use host cells and Chlamydia free of mycoplasma contamination for the analysis of Chlamydia-induced prostaglandin production.

During persistent infection, the intracellular bacterial pathogen Chlamydia trachomatis is viable but severely attenuates the production of new, infectious elementary bodies (EBs). To investigate the reasons for this lack of new EB output, we analysed the expression of chlamydial genes encoding products required for DNA replication and cell division, using in vitro models of active versus persistent infection and synovial tissue samples from patients with chronic Chlamydia-associated arthritis. Hep-2 cells were infected with K serovar C. trachomatis and harvested at t = 0-48 h post-infection (p.i; active). Human monocytes were infected similarly and harvested at t = 1-7 days p.i. (persistent). RNA preparations from infected/uninfected cells and patient samples were subjected to reverse transcription-polymerase chain reaction (RT-PCR) targeting polA, dnaA, mutS and parB mRNA, related to chlamydial DNA replication/segregation; these were expressed in infected Hep-2 cells from 11 to 48 h p.i; ftsK and ftsW, related to cell division, were expressed similarly. Real-time PCR analyses demonstrated that significant accumulation of chlamydial chromosome began at about 12 h p.i. in infected Hep-2 cells. In infected human monocytes, polA, dnaA, mutS and parB mRNA were produced from days 1-7 p.i. and were weakly expressed in patient samples. Real-time PCR indicated the continuing accumulation of chlamydial chromosome during the 7 day monocyte infection, although the rate of such accumulation was lower than that occurring during active growth. However, transcripts from ftsK and ftsW were detected only at 1 day p.i. in infected monocytes but not thereafter, and they were absent in all patient samples. Thus, genes whose products are required for chlamydial DNA replication are expressed during persistence, but transcription of genes whose products are required for cytokinesis is severely downregulated. These data explain, at least in part, the observed attenuation of new EB production during chlamydial persistence.

Several strains of Chlamydia trachomatis (CT) and C. pneumoniae (CP) from different sources were screened for mycoplasma contamination using a sensitive nested 16S rDNA polymerase chain reaction-specific for a broad range of mycoplasma species. Five of nine CT and 5/16 CP isolates were contaminated by mycoplasma. Mycoplasma fermentans, M. hyorhinis and M. hominis were found as contaminating agents. To our knowledge no data are available on whether coinfection of chlamydia with mycoplasma alters the biological behavior of chlamydia. Analysis of the biological effect of mycoplasma on chlamydial infection showed a profound mycoplasma-induced reduction of chlamydial growth. Mycoplasma were efficiently eliminated from chlamydial cultures in HEp-2 cells by treatment with mupirocin without affecting chlamydial replication or host cell growth. Two chlamydial strains, C. trachomatis serovar K and one clinical isolate of C. pneumoniae were purged by this method.