Chlamydia trachomatis is an obligate intracellular Gram-negative pathogen and the etiologic agent of significant ocular and genital tract diseases. Chlamydiae primarily infect epithelial cells, and the inflammatory response of these cells to the infection directs both the innate and adaptive immune response. This study focused on determining the cellular immune receptors involved in the early events following infection with the L2 serovar of C. trachomatis. We found that dominant negative MyD88 inhibited interleukin-8 (IL-8) secretion during a productive infection with chlamydia. Furthermore, expression of Toll-like receptor (TLR)-2 was required for IL-8 secretion from infected cells, whereas the effect of TLR4/MD-2 expression was minimal. Cell activation was dependent on infection with live, replicating bacteria, because infection with UV-irradiated bacteria and treatment of infected cells with chloramphenicol, but not ampicillin, abrogated the induction of IL-8 secretion. Finally, we show that both TLR2 and MyD88 co-localize with the intracellular chlamydial inclusion, suggesting that TLR2 is actively engaged in signaling from this intracellular location. These data support the role of TLR2 in the host response to infection with C. trachomatis. Our data further demonstrate that TLR2 and the adaptor MyD88 are specifically recruited to the bacterial or inclusion membrane during a productive infection with chlamydia and provide the first evidence that intracellular TLR2 is responsible for signal transduction during infection with an intracellular bacterium.Chlamydia trachomatis is an obligate intracellular bacterial pathogen associated with a variety of human diseases. The species of C. trachomatis can be divided into biovars, such as the trachoma biovar and the lymphogranuloma venereum biovar; these are further divided into serovars. In the developing world, serovars A-C are associated with trachoma, one of the oldest diseases caused by C. trachomatis infection, and the leading cause of preventable blindness in the world (reviewed in Ref. 1). Serovars D-K are also of the trachoma biovar and are all associated with genital tract disease as well as conjunctivitis and infant pneumonia. The World Health Organization reported 89 million new cases of genital Chlamydia infections in 1995, demonstrating the extent of this infection worldwide and hinting at the economic burden it has on healthcare (2). In 2003, 877,478 cases of infection with C. trachomatis were reported to the CDC from 50 states and the District of Columbia (3), making it the most common bacterial sexually transmitted infection. The lymphogranuloma venereum (LGV) 2 serovars L1, L2, and L3 are also sexually transmitted but are associated with a more invasive disease known as lymphogranuloma venereum (4). Although LGV is uncommon in the developed world, there have been recent outbreaks of LGV proctitis in the Netherlands and Western Europe (5, 6).The unique developmental cycle of the chlamydiae sets them apart from other bacterial species. C. trachomatis ...

Increased BH(4) by cardiomyocyte-specific overexpression of GTPCH-1 preserves the ability of IPC to elicit myocardial and mitochondrial protection that is impaired by HG, and this action appears to be dependent on NO.

Background-There remains no reliable noninvasive method to detect cardiac transplant rejection. Recently, speckle-tracking 2-dimensional strain echocardiography was shown to be sensitive in early detection of myocardial dysfunction in various models of cardiomyopathy. We aim to determine if 2DSE derived functional indices can detect cardiac transplant rejection.

Adult rat cardiac myocytes typically display a phenotypic response to cytokines manifested by low or no increases in nitric oxide (NO) production via inducible NO synthase (iNOS) that distinguishes them from other cell types. To better characterize this response, we examined the expression of tetrahydrobiopterin (BH4)-synthesizing and arginine-utilizing genes in cytokine-stimulated adult cardiac myocytes. Intracellular BH4 and 7,8-dihydrobiopterin (BH2) and NO production were quantified. Cytokines induced GTP cyclohydrolase and its feedback regulatory protein but with deficient levels of BH4 synthesis. Despite the induction of iNOS protein, cytokine-stimulated adult cardiac myocytes produced little or no increase in NO versus unstimulated cells. Western blot analysis under nonreducing conditions revealed the presence of iNOS monomers. Supplementation with sepiapterin (a precursor of BH4) increased BH4 as well as BH2, but this did not enhance NO levels or eliminate iNOS monomers. Similar findings were confirmed in vivo after treatment of rat cardiac allograft recipients with sepiapterin. It was found that expression of dihydrofolate reductase, required for full activity of the salvage pathway, was not detected in adult cardiac myocytes. Thus, adult cardiac myocytes have a limited capacity to synthesize BH4 after cytokine stimulation. The mechanisms involve posttranslational factors impairing de novo and salvage pathways. These conditions are unable to support active iNOS protein dimers necessary for NO production. These findings raise significant new questions about the prevailing understanding of how cytokines, via iNOS, cause cardiac dysfunction and injury in vivo during cardiac inflammatory disease states since cardiac myocytes are not a major source of high NO production.