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Persistence, more recently termed the chlamydial stress response, is a viable but non-infectious state constituting a divergence from the characteristic chlamydial biphasic developmental cycle. Damage/danger associated molecular patterns (DAMPs) are normal intracellular components or metabolites that, when released from cells, signal cellular damage/lysis. Purine metabolite DAMPs, including extracellular ATP and adenosine, inhibit chlamydial development in a species-specific manner. Viral co-infection has been shown to reversibly abrogate Chlamydia inclusion development, suggesting persistence/chlamydial stress. Because viral infection can cause host cell DAMP release, we hypothesized DAMPs may influence chlamydial development. Therefore, we examined the effect of extracellular ATP, adenosine, and cyclic AMP exposure, at 0 and 14 hours post infection, on C. pecorum and C. trachomatis serovar E development. In the absence of de novo host protein synthesis, exposure to DAMPs immediately post or at 14 hours post infection reduced inclusion size; however, the effect was less robust upon 14 hours post infection exposure. Additionally, upon exposure to DAMPs immediately post infection, bacteria per inclusion and subsequent infectivity were reduced in both Chlamydia species. These effects were reversible, and C. pecorum exhibited more pronounced recovery from DAMP exposure. Aberrant bodies, typical in virus-induced chlamydial persistence, were absent upon DAMP exposure. In the presence of de novo host protein synthesis, exposure to DAMPs immediately post infection reduced inclusion size, but only variably modulated chlamydial infectivity. Because chlamydial infection and other infections may increase local DAMP concentrations, DAMPs may influence Chlamydia infection in vivo, particularly in the context of poly-microbial infections. [ABSTRACT FROM AUTHOR]

Studies indicate that estrogen enhances Chlamydia trachomatis serovar E infection in genital epithelial cells. Hormones have direct and indirect effects on endometrial epithelial cells. Estrogen and progesterone exposure induces endometrial stromal cells to release effectors that subsequently regulate growth and maturation of uterine epithelial cells. Estrogen enhances C. trachomatis infection by aiding entry and intracellular development in endometrial epithelial cell (Ishikawa, IK)/SHT-290 stromal cell co-culture. Enhanced chlamydial infection was mediated by direct estrogen-stimulated signaling events in epithelial cells and indirectly via estrogen-induced stromal cell effectors. The current study investigates the effects of hormones on chlamydial development using culture conditions representative of the menstrual cycle. Chlamydia trachomatis-infected IK or IK/SHT-290 cultures were exposed to 10-8 M estrogen (E2), 10-7 M progesterone (P4) or a combination of both hormones (10-8 M E2 followed by 10-9 M E2/10-7 M P4). Chlamydial infectivity and progeny production were significantly decreased (30-66%) in cultures exposed to progesterone or estrogen/progesterone combination compared to estrogen alone. Thus, progesterone antagonized the positive effects of estrogen on chlamydial infection. These data indicate the susceptibility of endometrial epithelial cells to C. trachomatis infection during the menstrual cycle is altered by phase specific actions of sex hormones in the genital tract. [ABSTRACT FROM AUTHOR]

Abstract: Nectin-1 is an adhesion protein implicated in the organization of adherens junctions and tight junctions in epithelial cells. Previous studies in our laboratory demonstrated that nectin-1 accumulation was significantly decreased in Chlamydia trachomatis-infected HeLa cells. In the present study, Western blot analyses indicated that nectin-1 down-regulation was C. trachomatis concentration-dependent. The half-life of nectin-1 was also greatly diminished in C. trachomatis-infected cells compared to that observed in mock-infected cells, indicating that nectin-1 was likely down-regulated post-translationally. The chlamydia-secreted protease CPAF is known to degrade several important host proteins; CPAF expression within infected cells correlated with the time-dependent cleavage of nectin-1. Notably, CPAF proteolytic activity is inhibited by lactacystin but not by the proteosome inhibitor MG132. In vivo inhibition experiments demonstrated that nectin-1 down-regulation was blocked by lactacystin exposure. In contrast, MG132 had no effect. Finally, cell-free cleavage assays demonstrated that functional recombinant GST-CPAFwt protein degrades nectin-1. This degradation was blocked by lactacystin, as previously observed in vivo. Collectively, these results indicate that nectin-1 is degraded by CPAF in C. trachomatis-infected cells, a novel strategy that chlamydiae may use to aid their dissemination. [Copyright &y& Elsevier]

The Caprine Arthritis Encephalitis Virus (CAEV) genome encodes three structural (gag, pol, and env) and three accessory (rev, tat, and vif) genes. The Rev-C protein regulates Gag, Pol and Env expression by transporting their mRNAs to the cytoplasm. Rev trans-activation requires binding of Rev to an RNA structure called the Rev Response Element (RRE-C). Previous mutational analyses have shown that two domains of Rev are required for its function. The basic domain mediates RRE binding and multimer formation, and the nuclear export signal (NES) mediates trans-activation. Preliminary experiments demonstrate that Rev-C N-terminal deletion mutants bind the RRE less avidly than does wildtype Rev. As a result, it was hypothesized that an additional domain located in the N-terminal exon of Rev-C was required for optimal RRE binding. To test this hypothesis, Rev-C alanine scanning mutants were generated and in vitro RRE binding assays were performed. Alteration of Rev-C amino acids K13, E14, N15, V19, T20, M21 and R27 dramatically decreased affinity for RRE-C. These data demonstrate that Rev-C N-terminal amino acids are required for optimal RRE-C binding and suggest that a third functional domain exists within the N-terminus of Rev-C. [Copyright &y& Elsevier]

Caprine arthritis encephalitis virus (CAEV) is a lentivirus that is closely related to visna virus and more distantly related to the human lentivirus, Human Immunodeficiency Virus type 1 (HIV-1). The CAEV genome contains several small open reading frames (ORFs) that encode viral regulatory proteins. One of these non-structural proteins, Rev-C, is required for cytoplasmic transport of viral un/incompletely spliced mRNAs and efficient viral replication. In HIV-1 and visna virus, Rev is responsible for the temporal shift from non-structural protein synthesis to synthesis of structural proteins that is observed during the viral infectious cycle. Since it encodes a Rev protein, CAEV would be predicted to exhibit a similar temporal shift in gene expression during its replicative cycle. Immunoprecipitation analysis of 35S-pulse labeled, CAEV-infected goat synovial membrane (GSM) cells indicates that Rev-C is more abundant than is Gag at 12 h post-infection (PI); at later times PI Gag predominates. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) experiments using nuclear and cytoplasmic RNA from CAEV-infected GSM cells indicates that the viral unspliced gag mRNA accumulates significantly in the cytoplasm only after Rev is detected. These data indicate that a temporal shift from viral non-structural to structural gene expression occurs in CAEV infected GSM cells. [Copyright &y& Elsevier]

Focuses on a study which assessed the role of neurokinin-1 receptors in mediating the ganglion actions of substance P (SP) in rats. Details on SP; Materials and methods; Results of the study.

Examines increased ganglionic responses to substance P in hypertensive rats due to upregulation of neurokinin-1 receptors. Renal nerve, blood pressure and heart rate responses; RNA expression of neurokinin-1 receptors; Possible explanations for greater neurokinin-1 mRNA expression in hypertensive rats.

Altered gut microbial diversity has been associated with several chronic disease states, including heart failure. Stimulation of the vagus nerve, which innervates the heart and abdominal organs, is proving to be an effective therapeutic in heart failure. We hypothesized that cervical vagus nerve stimulation (VNS) could alter fecal flora and prevent aberrations observed in fecal samples from heart failure animals. To determine whether microbial abundances were altered by pressure overload (PO), leading to heart failure and VNS therapy, a VNS pulse generator was implanted with a stimulus lead on either the left or right vagus nerve before creation of PO by aortic constriction. Animals received intermittent, open-loop stimulation or sham treatment, and their heart function was monitored by echocardiography. Left ventricular end-systolic and diastolic volumes, as well as cardiac output, were impaired in PO animals compared with baseline. VNS mitigated these effects. Metagenetic analysis was then performed using 16S rRNA sequencing to identify bacterial genera present in fecal samples. The abundance of 10 genera was significantly altered by PO, 8 of which were mitigated in animals receiving either left- or right-sided VNS. Metatranscriptomics analyses indicate that the abundance of genera that express genes associated with ATP-binding cassette transport and amino sugar/nitrogen metabolism was significantly changed following PO. These gut flora changes were not observed in PO animals subjected to VNS. These data suggest that VNS prevents aberrant gut flora following PO, which could contribute to its beneficial effects in heart failure patients. [ABSTRACT FROM AUTHOR]

The article reports on the Yersinia pestis, a causative agent of bubonic plague, excrete L-aspartic acid at the expense of exogenous L-glutamic acid during expression of the low-calcium response. It discusses that the results of enzymic analysis provided suggest that a previously defined deficiency of aspartese (AspA) accounts for the phenomenon rather than an elevated oxaloacetate pool. It infers that the only known distinction between most sequenced isolates of aspA from Y. A study on the Yersinia pestis is presented.

Petunia hybrida and Citrus paradisi have significantly different flavonoid accumulation patterns. Petunia sp. tend to accumulate flavonol glycosides and anthocyanins while Citrus paradisi is known for its accumulation of flavanone diglycosides. One possible point of regulation of flavanone metabolism is flavanone 3-hydroxylase (F3H) expression. To test whether this is a key factor in the different flavanone usage by Petunia hybrida and Citrus paradisi, F3H mRNA expression in seedlings of different developmental stages was measured using semi-quantitative RT-PCR. Primers were designed to conserved regions of F3H and used to amplify an approximately 350 bp segment for quantitation by PhosphorImaging. Primary leaves of 32 day old grapefruit seedlings and a grapefruit flower bud had the highest levels of F3H mRNA expression. Petunia seedlings had much lower levels of F3H mRNA expression relative to grapefruit. The highest expression in petunia was in primary leaves and roots of 65 day old seedlings. These results indicate that preferential use of naringenin for production of high levels of flavanone glycosides in young grapefruit leaves cannot be attributed to decreased F3H mRNA expression. [Copyright &y& Elsevier]

Chlamydia trachomatis is the most common bacterial sexually transmitted pathogen, but more than 70% of patients fail to seek treatment due to the asymptomatic nature of these infections. Women suffer from numerous complications from chronic chlamydial infections, which include pelvic inflammatory disease and infertility. We previously demonstrated in culture that host cell nectin-1 knockdown significantly reduced chlamydial titers and inclusion size. Here, we sought to determine whether nectin-1 was required for chlamydial development in vivo by intravaginally infecting nectin-1-/- mice with Chlamydia muridarum and monitoring chlamydial shedding by chlamydial titer assay. We observed a significant reduction in chlamydial shedding in female nectin-1-/- mice compared to nectin-1+/+ control mice, an observation that was confirmed by PCR. Immunohistochemical staining in mouse cervical tissue confirmed that there are fewer chlamydial inclusions in Chlamydia-infected nectin-1-/- mice. Notably, anorectal chlamydial infections are becoming a substantial health burden, though little is known regarding the pathogenesis of these infections. We therefore established a novel male murine model of rectal chlamydial infection, which we used to determine whether nectin-1 is required for anorectal chlamydial infection in male mice. In contrast to the data from vaginal infection, no difference in rectal chlamydial shedding was observed when male nectin-1+/+ and nectin-1-/- mice were compared. Through the use of these two models, we have demonstrated that nectin-1 promotes chlamydial infection in the female genital tract but does not appear to contribute to rectal infection in male mice. These models could be used to further characterize tissue and sex related differences in chlamydial infection. [ABSTRACT FROM AUTHOR]

Chlamydia trachomatis and Herpes Simplex Virus-2 (HSV-2) genital tract co-infections have been reported in humans and studied in vitro but the clinical consequences are unknown. Limited epidemiologic evidence suggests that these co-infections could be more severe than single infections of either pathogen, but the host-pathogen interactions during co-infection remain uncharacterized. To determine whether disease progression and/or pathogen shedding differs between singly-infected and super-infected animals, we developed an in vivo super-infection model in which female BALB/c mice were vaginally infected with Chlamydia muridarum (Cm) followed later by HSV-2. Pre-infection with Chlamydia 3 or 9 days prior to HSV-2 super-infection conferred significant protection from HSV-2-induced neurologic disease and significantly reduced viral recovery compared to HSV-2 singly-infected controls. Neither protection from mortality nor reduced viral recovery were observed when mice were i) super-infected with HSV-2 on day 27 post Cm; ii) infected with UV-irradiated Cm and super-infected with HSV-2; or iii) azithromycin-treated prior to HSV-2 super-infection. Therefore, protection from HSV-2-induced disease requires active infection with viable chlamydiae and is not observed after chlamydial shedding ceases, either naturally or due to antibiotic treatment. Thus, Chlamydia-induced protection is transient and requires the continued presence of chlamydiae or their components. These data demonstrate that chlamydial pre-infection can alter progression of subsequent HSV-2 infection, with implications for HSV-2 transmission from co-infected humans. [ABSTRACT FROM AUTHOR]

The article discusses a study which showed that herpes simplex virus co-infection-induced Chlamydia trachomatis persistence is not mediated by any known persistence inducer or anti-chlamydial pathway. A tissue-culture model of Chlamydia trachomatis/herpes simplex virus type-2 (HSV-2) co-infection indicates that viral co-infection stimulates the formation of persistent chlamydiae. Luminex assays indicate that interferon (IFN)-gamma, interferon-alpha and tumor necrosis factor (TNF)-alpha are not released from co-infected cells while semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) studies show that IFN-beta, IFN-gamma, indoleamine 2,3-dioxygenase, lymphotoxin-alpha and inducible persistence is not stimulated by any persistence-associated cytokine.

Epidemiological studies have demonstrated that co-infections of herpes simplex virus type 2 (HSV-2) and Chlamydia trachomatis occur in vivo. Data from a tissue culture model of C. trachomatis/HSV-2 co-infection indicate that viral co-infection stimulates the formation of persistent chlamydiae. Transmission electron microscopic (TEM) analyses demonstrated that in both HeLa and HEC-1B cells, co-infection caused developing chlamydiae to exhibit swollen, aberrantly shaped reticulate bodies (RBs), characteristically observed in persistence. Additionally, HSV-2 co-infection suppressed production of infectious chlamydial elementary bodies (EBs) in both host cell types. Co-infection with HSV type 1 (HSV-1) produced similar morphologic alterations and abrogated infectious EB production. These data indicate that virus-induced chlamydial persistence was neither host cell- nor virus strain-specific. Purification of crude HSV-2 stocks demonstrated that viral particles were required for coinfection-induced chlamydial persistence to occur. Finally, co-infection with either UV-inactivated, replication-incompetent virus or replication-competent HSV-2 in the presence of cyclohexamide reduced chlamydial infectivity without altering chlamydial genomic DNA accumulation. These data demonstrate that productive viral replication is not required for the induction of chlamydial persistence and suggest that HSV attachment and entry can provide the necessary stimulus to alter C. trachomatis development. [ABSTRACT FROM AUTHOR]

Epidemiological and clinical studies have shown that double infection with herpes simplex virus type 2 (HSV-2) and Chlamydia trachomatis occurs in vivo. We hypothesized that co-infection would alter replication of these agents. To test this hypothesis, HeLa cells were infected with C. trachomatis serovar E, followed 24 h later by HSV-2 strain 333. Transmission electron microscopic (TEM) analyses indicated that, by 10 h after HSV addition, reticulate bodies (RBs) in co-infected cells were swollen, aberrantly shaped and electron-lucent. In infectious titre assays, HSV-2 co-infection abrogated production of infectious chlamydial progeny. Western blot analyses indicated that accumulation of chlamydial major outer membrane protein (MOMP) was decreased by HSV co-infection while accumulation of chlamydial heat-shock protein 60-1 (HSP60-1) was increased. Polymerase chain reaction (PCR) experiments indicated that chlamydial genome copy number was unaltered by HSV-2 superinfection. Semi-quantitative, reverse transcription PCR (RT-PCR) experiments demonstrated that levels of chlamydial groEL, ftsK, ftsW, dnaA and unprocessed 16S rRNA transcripts were not changed by HSV-2 super-infection. These data indicate that HSV-2 superinfection drives chlamydia into a viable but non-cultivable state, which is the hallmark of persistence. Because chlamydial HSP60-1 has been associated with immunopathology in vivo, these results also suggest that disease severity might be increased in co-infected individuals. [ABSTRACT FROM AUTHOR]

Chlamydia trachomatis /HSV-2 vaginal co-infections are seen clinically, suggesting that these sexually transmitted pathogens may interact. We previously established an intravaginal Chlamydia muridarum /HSV-2 super-infection model and observed that chlamydial pre-infection protects mice from a subsequent lethal HSV-2 challenge. However, the mechanism of protection remains unknown. The type I interferon, IFN-β, binds to the type I interferon receptor (IFNR), elicits a host cellular antiviral response and inhibits HSV replication in vitro and in vivo. Previous studies have demonstrated that C. muridarum infection stimulates genital tract (GT) IFN-β production; therefore, we hypothesized that chlamydial pre-infection protects mice from HSV-2 challenge via the IFN-β/IFNR-induced antiviral response. To test this prediction, we quantified IFN-β levels in vaginal swab samples. Detection of IFN-β in C. muridarum singly infected, but not in mock-infected animals, prompted the use of the super-infection model in IFNR knockout (IFNR−/−) mice. We observed that C. muridarum pre-infection reduces HSV-2-induced mortality by 40% in wild-type mice and by 60% IFNR−/− mice. Severity of HSV-2 disease symptoms and viral shedding was also similarly reduced by C. muridarum pre-infection. These data indicate that, while chlamydial infection induces GT production of IFN-β, type I IFN-induced antiviral responses are likely not required for the observed protective effect. [ABSTRACT FROM AUTHOR]