Broad-spectrum in vitro activity of macrophage infectivity potentiator inhibitors against Gram-negative bacteria and Leishmania major.

BACKGROUND: The macrophage infectivity potentiator (Mip) protein, which belongs to the immunophilin superfamily, is a peptidyl-prolyl cis/trans isomerase (PPIase) enzyme. Mip has been shown to be important for virulence in a wide range of pathogenic microorganisms. It has previously been demonstrated that small-molecule compounds designed to target Mip from the Gram-negative bacterium Burkholderia pseudomallei bind at the site of enzymatic activity of the protein, inhibiting the in vitro activity of Mip. OBJECTIVES: In this study, co-crystallography experiments with recombinant B. pseudomallei Mip (BpMip) protein and Mip inhibitors, biochemical analysis and computational modelling were used to predict the efficacy of lead compounds for broad-spectrum activity against other pathogens. METHODS: Binding activity of three lead compounds targeting BpMip was verified using surface plasmon resonance spectroscopy. The determination of crystal structures of BpMip in complex with these compounds, together with molecular modelling and in vitro assays, was used to determine whether the compounds have broad-spectrum antimicrobial activity against pathogens. RESULTS: Of the three lead small-molecule compounds, two were effective in inhibiting the PPIase activity of Mip proteins from Neisseria meningitidis, Klebsiella pneumoniae and Leishmania major. The compounds also reduced the intracellular burden of these pathogens using in vitro cell infection assays. CONCLUSIONS: These results indicate that Mip is a novel antivirulence target that can be inhibited using small-molecule compounds that prove to be promising broad-spectrum drug candidates in vitro. Further optimization of compounds is required for in vivo evaluation and future clinical applications.

Evaluation of VT-1161 for Treatment of Coccidioidomycosis in Murine Infection Models.

Coccidioidomycosis, or valley fever, is a growing health concern endemic to the southwestern United States. Safer, more effective, and more easily administered drugs are needed especially for severe, chronic, or unresponsive infections. The novel fungal CYP51 inhibitor VT-1161 demonstrated in vitro antifungal activity, with MIC50 and MIC90 values of 1 and 2 µg/ml, respectively, against 52 Coccidioides clinical isolates. In the initial animal study, oral doses of 10 and 50 mg/kg VT-1161 significantly reduced fungal burdens and increased survival time in a lethal respiratory model in comparison with treatment with a placebo (P < 0.001). Oral doses of 25 and 50 mg/kg VT-1161 were similarly efficacious in the murine central nervous system (CNS) model compared to placebo treatment (P < 0.001). All comparisons with the positive-control drug, fluconazole at 50 mg/kg per day, demonstrated either statistical equivalence or superiority of VT-1161. VT-1161 treatment also prevented dissemination of infection from the original inoculation site to a greater extent than fluconazole. Many of these in vivo results can be explained by the long half-life of VT-1161 leading to sustained high plasma levels. Thus, the efficacy and pharmacokinetics of VT-1161 are attractive characteristics for long-term treatment of this serious fungal infection.

Quality control within the multicentre perfusion CT study of primary colorectal cancer (PROSPeCT): results of an iodine density phantom study.

OBJECTIVES: To assess the cross-centre consistency of iodine enhancement, contrast-to-noise ratio and radiation dose in a multicentre perfusion CT trial of colorectal cancer. MATERIALS AND METHODS: A cylindrical water phantom containing different iodine inserts was examined on seven CT models in 13 hospitals. The relationship between CT number (Hounsfield units, HU) and iodine concentration (milligrams per millilitre) was established and contrast-to-noise ratios (CNRs) calculated. Radiation doses (CTDIvol, DLP) were compared across all sites. RESULTS: There was a linear relationship between CT number and iodine density. Iodine enhancement varied by a factor of at most 1.10, and image noise by at most 1.5 across the study sites. At an iodine concentration of 1 mg ml(-1) and 100 kV, CNRs ranged from 3.6 to 4.8 in the 220-mm phantom and from 1.4 to 1.9 in the 300-mm phantom. Doses varied by a factor of at most 2.4, but remained within study dose constraints. Iterative reconstruction algorithms did not alter iodine enhancement but resulted in reduced image noise by a factor of at most 2.2, allowing a potential dose decrease of at most 80% compared to filtered back projection (FBP). CONCLUSIONS: Quality control of CT performance across centres indicates that CNR values remain relatively consistent across all sites, giving acceptable image quality within the agreed dose constraints. KEY POINTS: Quality control is essential in a multicentre setting to enable CT quantification. CNRs in a body-sized phantom had the recommended value of at least 1.5. CTDIs and DLPs varied by factors of 1.8 and 2.4 respectively.

Chlamydia trachomatis infection results in a modest pro-inflammatory cytokine response and a decrease in T cell chemokine secretion in human polarized endocervical epithelial cells.

The endocervical epithelium is a major reservoir for Chlamydia trachomatis in women, and genital infections are extended in their duration. Epithelial cells act as mucosal sentinels by secreting cytokines and chemokines in response to pathogen challenge and infection. We therefore determined the signature cytokine and chemokine response of primary-like endocervix-derived epithelial cells in response to a common genital serovar (D) of C. trachomatis. For these studies, we used a recently-established polarized, immortalized, endocervical epithelial cell model (polA2EN) that maintains, in vitro, the architectural and functional characteristics of endocervical epithelial cells in vivo including the production of pro-inflammatory cytokines. PolA2EN cells were susceptible to C. trachomatis infection, and chlamydiae in these cells underwent a normal developmental cycle as determined by a one-step growth curve. IL1α protein levels were increased in both apical and basolateral secretions of C. trachomatis infected polA2EN cells, but this response did not occur until 72h after infection. Furthermore, protein levels of the pro-inflammatory cytokines and chemokines IL6, TNFα and CXCL8 were not significantly different between C. trachomatis infected polA2EN cells and mock infected cells at any time during the chlamydial developmental cycle up to 120h post-infection. Intriguingly, C. trachomatis infection resulted in a significant decrease in the constitutive secretion of T cell chemokines IP10 and RANTES, and this required a productive C. trachomatis infection. Examination of anti-inflammatory cytokines revealed a high constitutive apical secretion of IL1ra from polA2EN cells that was not significantly modulated by C. trachomatis infection. IL-11 was induced by C. trachomatis, although only from the basolateral membrane. These results suggest that C. trachomatis can use evasion strategies to circumvent a robust pro-inflammatory cytokine and chemokine response. These evasion strategies, together with the inherent immune repertoire of endocervical epithelial cells, may aid chlamydiae in establishing, and possibly sustaining, an intracellular niche in microenvironments of the endocervix in vivo.

The major CD8 T cell effector memory subset in the normal and Chlamydia trachomatis-infected human endocervix is low in perforin.

BACKGROUND: The local tissue microenvironment plays an important role in the induction, homing, maintenance and development of effector functions of T cells. Thus, site-specific differences in phenotypes of mucosal and systemic T cell populations have been observed. Chlamydia trachomatis most commonly infects the endocervix in women, yet little is known about Chlamydia-specific effector T cell immunity at this unique mucosal site. Our previous flow-cytometry-based study of cervical-cytobrush retrieved cells indicated that CD8 T cells are significantly increased in the C. trachomatis-infected human endocervix. The cytolytic function of CD8 T cells is important in the protective immunity against many intracellular pathogens, and requires the cytolytic granule perforin to facilitate the entry of other molecules that mediate the lysis of target cells. Determination of perforin expression of the CD8 T cell population in the endocervix would therefore provide insights on the granule-mediated cytolytic potential of these cells at this site. RESULTS: Our histological data revealed that C. trachomatis-infected tissues have significantly higher numbers of CD3 and CD8 T cells compared to non-infected tissues (p<0.01), and that the majority of CD8+ cells do not express perforin in situ. A subsequent flow cytometric analysis of paired blood and endocervix-derived cells (n=16) revealed that while all the CD8 T cell subsets: naïve, effector memory (TEM), central memory (TCM) and terminally differentiated effector memory (TEMRA) can be found in the blood, the endocervix is populated mainly by the TEM CD8 T cell subset. Our data also showed that perforin expression in the TEM population is significantly lower in the endocervix than in the blood of C. trachomatis positive women (n=15; p<0.0001), as well as in C. trachomatis-negative individuals (n=6; p<0.05). Interestingly, our in vitro co-culture study suggests that the exposure of HeLa 229 cervical epithelial cells to IFN gamma could potentially induce a decrease in perforin content in CD8 TEM cells in the same microenvironment. CONCLUSIONS: The low perforin content of CD8 TEM cells in the endocervix, the local site of C. trachomatis infection in women, may reflect the unique immunological environment that balances immune protection against sexually transmitted infections and immune- tolerance to support conception.

Altered protein secretion of Chlamydia trachomatis in persistently infected human endocervical epithelial cells.

Chlamydia trachomatis is the most common bacterial infection of the human reproductive tract globally; however, the mechanisms underlying the adaptation of the organism to its natural target cells, human endocervical epithelial cells, are not clearly understood. To secure its intracellular niche, C. trachomatis must modulate the host cellular machinery by secreting virulence factors into the host cytosol to facilitate bacterial growth and survival. Here we used primary human endocervical epithelial cells and HeLa cells infected with C. trachomatis to examine the secretion of bacterial proteins during productive growth and persistent growth induced by ampicillin. Specifically, we observed a decrease in secretable chlamydial protease-like activity factor (CPAF) in the cytosol of host epithelial cells exposed to ampicillin with no evident reduction of CPAF product by C. trachomatis. In contrast, the expression of CopN and Tarp was downregulated, suggesting that C. trachomatis responds to ampicillin exposure by selectively altering the expression of secretable proteins. In addition, we observed a greater accumulation of outer-membrane vesicles from C. trachomatis in persistently infected cells. Taken together, these results suggest that the regulation of both gene expression and the secretion of chlamydial virulence proteins is involved in the adaptation of the bacteria to a persistent infection state in human genital epithelial cells.

A quantitative enzyme-linked immunoassay (ELISA) to approximate complement-fixing antibody titers in serum from patients with coccidioidomycosis.

Coccidioidomycosis is most frequently diagnosed serologically, and the quantitative test for complement-fixing antibodies is considered prognostically useful. Because complement-fixing antibody testing is complex, labor-intensive, and poorly standardized, an enzyme-linked immunoassay (ELISA) alternative would be attractive. In this report, we restrict the complement-fixing, antibody-binding domain to a 200-amino-acid recombinant peptide of the known antigen. Over-lapping truncations of this peptide do not bind complement-fixing antibodies, suggesting that the responsible epitope(s) are conformational. Further, anchoring the antigenic peptide to the ELISA plate by means of a C-terminal biotin-mimic peptide tag instead of allowing the peptide to randomly adhere to the plastic plate improves sensitivity of antibody detection by 1-2 logs in different sera. The newly developed ELISA shows a significant quantitative correlation with complement-fixing antibody titers. This ELISA shows potential as the basis for a new quantitative assay for coccidioidal antibodies.

Selecting a CT scanner for cardiac imaging: the heart of the matter.

Coronary angiography to assess the presence and degree of arterial stenosis is an examination now routinely performed on CT scanners. Although developments in CT technology over recent years have made great strides in improving the diagnostic accuracy of this technique, patients with certain characteristics can still be "difficult to image". The various groups will benefit from different technological enhancements depending on the type of challenge they present. Good temporal and spatial resolution, wide longitudinal (z-axis) detector coverage and high X-ray output are the key requirements of a successful CT coronary angiography (CTCA) scan. The requirement for optimal patient dose is a given. The different scanner models recommended for CTCA all excel in different aspects. The specification data presented here for these scanners and the explanation of the impact of the different features should help in making a more informed decision when selecting a scanner for CTCA.

Membrane vesicle production by Chlamydia trachomatis as an adaptive response.

Bacteria have evolved specific adaptive responses to cope with changing environments. These adaptations include stress response phenotypes with dynamic modifications of the bacterial cell envelope and generation of membrane vesicles (MVs). The obligate intracellular bacterium, Chlamydia trachomatis, typically has a biphasic lifestyle, but can enter into an altered growth state typified by morphologically aberrant chlamydial forms, termed persistent growth forms, when induced by stress in vitro. How C. trachomatis can adapt to a persistent growth state in host epithelial cells in vivo is not well understood, but is an important question, since it extends the host-bacterial relationship in vitro and has thus been indicated as a survival mechanism in chronic chlamydial infections. Here, we review recent findings on the mechanistic aspects of bacterial adaptation to stress with a focus on how C. trachomatis remodels its envelope, produces MVs, and the potential important consequences of MV production with respect to host-pathogen interactions. Emerging data suggest that the generation of MVs may be an important mechanism for C. trachomatis intracellular survival of stress, and thus may aid in the establishment of a chronic infection in human genital epithelial cells.

Morphologic and molecular evaluation of Chlamydia trachomatis growth in human endocervix reveals distinct growth patterns.

In vitro models of Chlamydia trachomatis growth have long been studied to predict growth in vivo. Alternative or persistent growth modes in vitro have been shown to occur under the influence of numerous stressors but have not been studied in vivo. Here, we report the development of methods for sampling human infections from the endocervix in a manner that permits a multifaceted analysis of the bacteria, host and the endocervical environment. Our approach permits evaluating total bacterial load, transcriptional patterns, morphology by immunofluorescence and electron microscopy, and levels of cytokines and nutrients in the infection microenvironment. By applying this approach to two pilot patients with disparate infections, we have determined that their contrasting growth patterns correlate with strikingly distinct transcriptional biomarkers, and are associated with differences in local levels of IFNγ. Our multifaceted approach will be useful to dissect infections in the human host and be useful in identifying patients at risk for chronic disease. Importantly, the molecular and morphological analyses described here indicate that persistent growth forms can be isolated from the human endocervix when the infection microenvironment resembles the in vitro model of IFNγ-induced persistence.

Health Protective Behaviors among Young People Living with HIV/AIDS.

Young people living with HIV/AIDS (YPLH) in the post-HAART era have the potential to manage their HIV as a chronic illness rather than as an almost inevitable terminal disease. However, little is known about behaviors YPLH can engage in to promote or protect health beyond taking anti-retroviral (ARV) medicines. The current study fills an important gap in existing research by identifying correlates of Health Protective Behaviors (HPB) within an urban sample of YPLH. Participants (n=134) were recruited from two pediatric clinics serving adolescents living with HIV who met the following criteria: HIV-infected and aware of their status, 13-24 years old, residing in the Washington, DC metropolitan area, currently prescribed ARV or due to begin ARV within 3 months, and able to understand and sign a written consent form. Each participant was interviewed via Audio computer-assisted self-interview (ACASI) and reported the percentage of possible time they engaged in six HPB during the prior month using a 10-point scale (e.g., 5=50%). Results indicate that most patients engaged in the measured health protective behaviors more than half the time, although patients 18 and older engaged in all HPB less frequently than their younger counterparts. Patients with adherence > 90% were far more likely to engage in HPB than those with poorer adherence. The age differences in HPB highlight a need for broader scaffolding in the transition to independent living and adult health care. Given the relationship between adherence and HPB, the establishment and maintenance of healthy lifestyle practices early in adolescence can translate into positive long-term health outcomes.

Innate immune mediator profiles and their regulation in a novel polarized immortalized epithelial cell model derived from human endocervix.

The endocervix in the female reproductive tract (FRT) is susceptible to sexually transmitted pathogens such as Chlamydia trachomatis and Neisseria gonorrhoeae. Endocervical epithelial cells in vivo make innate immune mediators that likely aid in the protection from these pathogens. In vitro studies to investigate the innate epithelial cell immune response to endocervical pathogens have been hindered by the paucity of human endocervix-derived epithelial cell lines that display the differentiation proteins and functional characteristics of their site of origin. We have established an immortalized epithelial cell line (A2EN) derived from an endocervical tissue explant that can be polarized to exhibit distinct apical and basolateral membrane domains. Polarized A2EN cells secrete mucus at their apical surface, and express MUC5B, a mucin specific to the endocervix. Polarized A2EN cells also express hormone receptors that respond appropriately to female steroid hormones. Polarized A2EN cells can be stimulated with the toll-like receptor 3 agonist, polyI:C, to express anti-microbial peptides (AMPs) as well as pro-inflammatory cytokines and chemokines. Cytokines and chemokines are also differentially secreted depending on the hormone milieu in which the cells are exposed. We conclude that polarized A2EN cells maintain distinctive phenotypic and functional characteristics of the epithelial cells found in the endocervix and, hence, could provide a useful, new in vitro model system for investigations on the role of endogenous and exogenous factors that regulate endocervical epithelial cell immunity including studies on sexually transmitted infections and topical microbicides.

Psychotropic medication patterns among youth in foster care.

CONTEXT: Studies have revealed that youth in foster care covered by Medicaid insurance receive psychotropic medication at a rate > 3 times that of Medicaid-insured youth who qualify by low family income. Systematic data on patterns of medication treatment, particularly concomitant drugs, for youth in foster care are limited. OBJECTIVE: The purpose of this work was to describe and quantify patterns of psychotropic monotherapy and concomitant therapy prescribed to a randomly selected, 1-month sample of youth in foster care who had been receiving psychotropic medication. METHODS. Medicaid data were accessed for a July 2004 random sample of 472 medicated youth in foster care aged 0 through 19 years from a southwestern US state. Psychotropic medication treatment data were identified by concomitant pattern, frequency, medication class, subclass, and drug entity and were analyzed in relation to age group; gender; race or ethnicity; International Classification of Diseases, Ninth Revision, psychiatric diagnosis; and physician specialty. RESULTS: Of the foster children who had been dispensed psychotropic medication, 41.3% received > or = 3 different classes of these drugs during July 2004, and 15.9% received > or = 4 different classes. The most frequently used medications were antidepressants (56.8%), attention-deficit/hyperactivity disorder drugs (55.9%), and antipsychotic agents (53.2%). The use of specific psychotropic medication classes varied little by diagnostic grouping. Psychiatrists prescribed 93% of the psychotropic medication dispensed to youth in foster care. The use of > or = 2 drugs within the same psychotropic medication class was noted in 22.2% of those who were given prescribed drugs concomitantly. CONCLUSIONS: Concomitant psychotropic medication treatment is frequent for youth in foster care and lacks substantive evidence as to its effectiveness and safety.