An in vitro cell culture system was used to study the effect ofinterferon V(IFN-y) on Chiamydia trachomatis growth and differentiation. The effect of IFN-y on chlamydiae was dose-dependent. IFN-y at 2 ng/ml completely inhibited chlamydial growth and differentiation; however, persistent infection was established when chlamydiae were cultured with IFN-y at 0.2 ng/ml. Persistent infection was characterized by the development of noninfectious atypical chlamydial forms from which infectious progeny could be recovered only when IFN-y was removed from the culture system. defined, but previous studies demonstrate that treatment of host cells with interferon 'y (IFN-'y) before infection with chlamydiae results in the formation of atypical RBs that fail to differentiate into infectious progeny (8-12). Because persistent infections have been proposed as a source of antigen for the stimulation of immunopathology that leads to blindness and infertility, we evaluated whether infectious chlamydiae could be rescued from the aberrant RB forms that follow IFN-y treatment and whether the levels of important chlamydial immunogens were altered during abnormal growth.MATERIALS AND METHODS Organism. C. trachomatis serovar A/Har-13 was grown in HeLa 229 cells, and EBs were purified by discontinuous density centrifugation in Renografin (Squibb) (13).Preparation of IFN-y-Treated Cultures and Assay for Infectivity. HeLa 229 cells in minimal essential medium with 10% fetal bovine serum (MEM-10) were plated at a density of 1.5 x 105 cells per well in 24-well culture plates and maintained at 37°C in 5% CO2. Eighteen to 24 hr later the cell monolayers were washed once with Hanks' balanced salt solution (HBSS) and then treated for 15 min at room temperature with HBSS containing DEAE-dextran (45 ,g/ml), followed by two washes with HBSS. The final wash was removed and replaced with 0.2 ml of 0.25 M sucrose/10 mM sodium phosphate/5 mM L-glutamic acid (SPG), pH 7.2, containing 3 x 105 inclusion-forming units (IFU) of C. trachomatis serovar A (HAR-13), and incubated for 2 hr at 37°C on a rocker platform. Two hours after infection the inoculum was removed and replaced with 0.5 ml of either MEM-10 or MEM-10 containing recombinant human IFN-y at 0.05, 0.2, or 2.0 ng/ml (Biogen). At the indicated times the monolayers were washed three times with HBSS, and cells were scraped from the culture dishes into 0.5 ml of SPG solution and frozen until all samples were collected. Samples were briefly sonicated to disrupt the HeLa cells and release the chlamydial EBs. The disrupted cell suspensions were diluted in SPG solution and used to inoculate fresh monolayers of HeLa cells, as described above. Infected monolayers were cultured in MEM-10 containing cycloheximide at 1 ,ug/ml for 48 hr, then washed once with HBSS, and fixed with methanol; inclusions were visualized by indirect immunofluorescence with an anti-major outer membrane protein (MOMP) monoclonal antibody (mAb) (A-20) (14,15).In experiments to determine whether infectious chlamydiae could be res...

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Recent appreciation of atherosclerosis as a chronic, inflammatory disease has rekindled efforts to examine the role that infectious agents may play in atherogenesis. In particular, much interest has focused on infection with Chlamydia pneumoniae. The possibility that a prokaryote contributes to atherogenesis has high clinical interest, as C pneumoniae infection may be a treatable risk factor. To review the evidence implicating C pneumoniae in the pathogenesis of atherosclerosis, we searched MEDLINE for articles published between January 1966 and October 2002 on the association of C pneumoniae and atherosclerosis. We also used online resources, texts, meeting abstracts, and expert opinion. We included 5 types of studies (epidemiological, pathology based, animal model, cell biology, and human antibiotic treatment trials) and extracted diagnostic, pathophysiologic, and therapeutic information from the selected literature; consensus was reached on interpretation discrepancies. Chlamydia pneumoniae is associated with atherosclerosis by epidemiological and pathology-based studies. Animal model and cell biology studies suggest that the pathogen can modulate atheroma biology, including lipid- and inflammatory-related processes. Although some preliminary antibiotic treatment trials in patients with coronary artery disease indicated a reduction in recurrent coronary events, larger studies have not shown benefits in individuals with stable coronary artery disease. It is unlikely that C pneumoniae infection is necessary to initiate atherosclerosis. Furthermore, conventional antibiotic therapy may not eradicate the organism or reduce mortality in individuals with atherosclerotic vascular disease. Nevertheless, the current body of evidence establishes this pathogen as a plausible, potentially modifiable risk factor in cardiovascular disease.

Interferon (IFN)-induced tryptophan degradation, catalyzed by indoleamine 2,3-dioxygenase (IDO), has been shown to mediate antimicrobial activity in epithelial cells. IDO activity has also been augmented in peripheral blood mononuclear cells (PBMC) treated with IFN or interleukin-2 (IL-2). The effector cells in this population have now been further characterized. PBMCs were isolated from normal donors, separated into monocyte and lymphocyte populations by plastic adherence, treated with IFN or IL-2, and cultivated in medium supplemented with [3H]tryptophan. Culture supernatants were collected after a 48-h incubation and fractionated by high-performance liquid chromatography; radioactivity was determined in fractions corresponding to tryptophan and its metabolites. IFN-gamma and IFN-beta induced IDO activity only in monocytes (plastic-adherent, nonspecific esterase-positive PBMCs). The induction of IDO activity by IL-2 required both monocytes and lymphocytes. Interaction was required between these populations for induction of IDO by IL-2, due to production of IFN-gamma by T lymphocytes, with subsequent IFN-gamma-mediated induction of IDO in monocytes. A number of myeloid cell lines as well as monocyte-derived macrophages were also tested for their ability to be induced to degrade tryptophan in response to IFN treatment. Monocyte-derived macrophages were found to retain their capacity to be induced by IFN-gamma and IFN-beta to degrade tryptophan after differentiation, and to possess seven times more IDO activity per cell than IFN-induced monocytes. However, the presence of lipopolysaccharide (LPS) in the culture medium was required for the maximum induction of IDO activity by IFN-beta. Furthermore, higher concentrations of LPS were sufficient to induce IDO activity in macrophages in the absence of exogenous IFN.

Foam cell formation is the hallmark of early atherosclerosis. It was found that the intracellular bacterium Chlamydia pneumoniae induces foam cell formation by human monocyte-derived macrophages. Exposure of macrophages to C. pneumoniae followed by low-density lipoprotein (LDL) caused a marked increase in the number of foam cells and accumulation of cholesteryl esters. Foam cell formation was not inhibited by the antioxidant butylated hydroxytoluene nor fucoidan, suggesting that lipid accumulation did not involve scavenger receptors. In contrast, addition of heparin, which blocks binding of LDL to the LDL receptor, inhibited C. pneumoniae-induced foam cell formation, suggesting that the pathogen induced lipid accumulation by dysregulating native LDL uptake or metabolism (or both). These data demonstrate that an infectious agent can induce macrophage foam cell formation and implicate C. pneumoniae as a causative factor in atherosclerosis.

SummaryThe developmentally regulated intracellular pathogen Chlamydia pneumoniae is a natural tryptophan auxotroph. These organisms survive tryptophan starvation induced by host cell activation with IFNg by blocking maturation to the infectious form. In most bacteria, the stringent response is induced during amino acid starvation to promote survival. However, the response of obligate intracellular pathogens, which are predicted to lack stringent responses to amino acid starvation, is poorly characterized. Chlamydial transcription and translation were analysed during IFNg-mediated tryptophan starvation using genomic normalization methods, and the data revealed the novel findings that: (i) global chlamydial transcription was upregulated; and (ii) protein synthesis was dramatically reduced. These results indicate a dysregulation of developmental gene expression and an uncoupling of transcription from translation. These observations represent an alternative survival strategy for host-adapted obligate intracellular bacterial pathogens that have lost the genes for stringent control during reductive evolution.

Human uroepithelial (T24) cells were incubated for 24 h in the presence of various concentrations of human recombinant gamma interferon (Hu-rIFN-y) and then infected with the 6BC strain of Chlamydia psittaci. This resulted in a reduction of intracellular chlamydial inclusion development in proportion to the concentration of Hu-rIFN-y present when Giemsa-stained cells were examined by light microscopy 24 h after infection. When tryptophan was added to Hu-rIFN-y-treated cells just after infection, reversal of the Hu-rIFN-y-mediated inhibition occurred in proportion to the concentration of tryptophan added. Addition of either isoleucine or lysine did not result in reversal of the antichlamydial state. Transport of L-[3H]tryptophan into acid-soluble intracellular pools was found to be greatly enhanced in Hu-rIFN-y-treated T24 cells compared with the rates measured for untreated cells. Transport of [3HJleucine was not increased in treated cells. Cells treated with Hu-rIFN-y also degraded L-[3H]tryptophan to catabolites that cochromatographed with N-formylkynurenine and kynurenine as measured by high-performance liquid chromatography. We conclude that Hu-rIFN-ymediated inhibition of intracellular C. psittaci replication in T24 cells occurs by depletion of the essential amino acid tryptophan, most likely via the induction of indoleamine-2,3-dioxygenase, the initial enzyme of tryptophan catabolism.

Previous studies have shown that the immune-regulated cytokine gamma interferon (IFN-fy) activates host cells to restrict intracellular growth of the bacterial pathogen Chlamydia trachomatis by induction of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). Recently, subinhibitory levels of IFN-y were used to generate an in vitro persistent chlamydial infection characterized by large aberrant, noninfectious reticulate bodies from which infectious progeny could be recovered following the removal of IFN-fy. Studies were done to determine if the mechanism functioning to induce chlamydiae to enter a persistent state in the presence of low levels of IFN-'y was similar to that reported to inhibit chlamydial growth. Host cells treated with levels of IFN-y required to induce persistence were assessed for IDO activity by high-performance liquid chromatography analysis of tryptophan and its catabolic products. Substantial tryptophan catabolism was detected in acid-soluble cellular pools, indicating that the intracellular availability of this essential amino acid was limited under these conditions. In addition, a mutant cell line responsive to IFN-'y but deficient in IDO activity was shown to support C. trachomatis growth, but aberrant organisms were not induced in response to IFN-y treatment. Analyses of infected cells cultured in medium with incremental levels of exogenous