Mycoplasma hominis impacts gene expression in Trichomonas vaginalis

Parasitology Research - In Europe, up to 90% of isolated Trichomonas vaginalis strains are naturally infected with Mycoplasma hominis, a facultative pathogen of the human genital tract. The...     

Phage Display-directed Discovery of LEDGF/p75 Binding Cyclic Peptide Inhibitors of HIV Replication

The interaction between the human immunodeficiency virus (HIV) integrase (IN) and its cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) is crucial for HIV replication. While recently discovered LEDGINs inhibit HIV-1 replication by occupying the LEDGF/p75 pocket in IN, it remained to be demonstrated whether LEDGF/p75 by itself can be targeted. By phage display we identified cyclic peptides (CPs) as the first LEDGF/p75 ligands that inhibit the LEDGF/p75–IN interaction. The CPs inhibit HIV replication in different cell lines without overt toxicity. In accord with the role of LEDGF/p75 in HIV integration and its inhibition by LEDGINs, CP64, and CP65 block HIV replication primarily by inhibiting the integration step. The CPs retained activity against HIV strains resistant to raltegravir or LEDGINs. Saturation transfer difference (STD) NMR showed residues in CP64 that strongly interact with LEDGF/p75 but not with HIV IN. Mutational analysis identified tryptophan as an important residue responsible for the activity of the peptides. Serial passaging of virus in the presence of CPs did not yield resistant strains. Our work provides proof-of-concept for direct targeting of LEDGF/p75 as novel therapeutic strategy and the CPs thereby serve as scaffold for future development of new HIV therapeutics.     

A subconvulsive dose of kainate selectively compromises astrocytic metabolism in the mouse brain in vivo

Despite the well-established use of kainate as a model for seizure activity and temporal lobe epilepsy, most studies have been performed at doses giving rise to general limbic seizures and have mainly focused on neuronal function. Little is known about the effect of lower doses of kainate on cerebral metabolism and particularly that associated with astrocytes. We investigated astrocytic and neuronal metabolism in the cerebral cortex of adult mice after treatment with saline (controls), a subconvulsive or a mildly convulsive dose of kainate. A combination of [1,2-¹³C]acetate and [1-¹³C]glucose was injected and subsequent nuclear magnetic resonance spectroscopy of cortical extracts was employed to distinctively map astrocytic and neuronal metabolism. The subconvulsive dose of kainate led to an instantaneous increase in the cortical lactate content, a subsequent reduction in the amount of [4,5-¹³C]glutamine and an increase in the calculated astrocytic TCA cycle activity. In contrast, the convulsive dose led to decrements in the cortical content and ¹³C labeling of glutamate, glutamine, GABA, and aspartate. Evidence is provided that astrocytic metabolism is affected by a subconvulsive dose of kainate, whereas a higher dose is required to affect neuronal metabolism. The cerebral glycogen content was dose-dependently reduced by kainate supporting a role for glycogen during seizure activity.     

Effectiveness of antiplatelet therapy in atherosclerotic disease: comparing the ASA low-response prevalence in CVD,CAD and PAD

Although acetylsalicylic acid (ASA, aspirin) reduces the risk of ischemic events in patients with atherosclerosis, a substantial number of incidents continue to occur. As only limited data exist we evaluated the antiplatelet effectiveness of ASA in patients with different manifestations of atherosclerosis as in cerebrovascular, coronary artery and peripheral arterial disease (CVD, CAD, PAD). For the evaluation of the antiplatelet effectiveness of ASA we used whole blood aggregometry (Chrono-log Model 590). The patients in the different subgroups received ASA 100, 200 or 500 mg daily. We analysed 737 consecutive patients: 47.5 % with CVD, 33.6 % with CAD, and 18.9 % with PAD. We identified 28.0 % of the CVD, 18.1 % of the CAD and 21.6 % of the PAD patients to be ASA low-responder (ALR). Comparing subgroups treated with 100 mg ASA, 36.4 % were ALR in the CVD group as were 13.1 % of the CAD and 21.6 % of the PAD patients. Multivariate regression analysis revealed an odds ratio for being ALR of 4.50 (95 % confidence interval (CI) 1.70–11.9) when 100 mg and of 2.97 (95 % CI 1.58–5.60) when 200 mg ASA was taken compared to a dose of 500 mg. Despite the proven benefits of antiplatelet therapy in the secondary prevention of atherosclerotic disease, current antiplatelet management is suboptimal as up to 36 % of patients failed to achieve an adequate platelet inhibitory effect. Our findings may explain, at least in part, the high rates of cardiovascular events observed in the course of atherothrombotic disease and support the need to improve antiplatelet therapy.     

Commentary: Dermal penetration of bisphenol A—Consequences for risk assessment

With this comment we would raise awareness for applying appropriate procedures in route-to-route extrapolation. The paper of Demierre et al. (2012) prompted us to comment on the simple approach for route-to-route extrapolation and to explain some short comings. For the risk assessment of exposures resulting from a non-oral route, route-to-route extrapolation is often done by correcting the non-oral route exposure by the route specific absorption into the systemic circulation and comparing the result with the (oral) threshold value. Making use of this procedure means that an internal dose obtained from the non-oral route is compared with an external dose of the oral route. This procedure would be appropriate only if the absorption on the oral route is 100%. If the absorption on the oral route is less than 100% the procedure may underestimate the risk of the exposure of the non-oral route. For some chemicals with a high first pass metabolism in the liver, e.g. BPA, the situation is even more complex and in addition, the target organ for toxicity has to be taken into consideration.     

The pentose phosphate pathway and pyruvate carboxylation after neonatal hypoxic-ischemic brain injury

The neonatal brain is vulnerable to oxidative stress, and the pentose phosphate pathway (PPP) may be of particular importance to limit the injury. Furthermore, in the neonatal brain, neurons depend on de novo synthesis of neurotransmitters via pyruvate carboxylase (PC) in astrocytes to increase neurotransmitter pools. In the adult brain, PPP activity increases in response to various injuries while pyruvate carboxylation is reduced after ischemia. However, little is known about the response of these pathways after neonatal hypoxia-ischemia (HI). To this end, 7-day-old rats were subjected to unilateral carotid artery ligation followed by hypoxia. Animals were injected with [1,2-¹³C]glucose during the recovery phase and extracts of cerebral hemispheres ipsi- and contralateral to the operation were analyzed using ¹H- and ¹³C-NMR (nuclear magnetic resonance) spectroscopy and high-performance liquid chromatography (HPLC). After HI, glucose levels were increased and there was evidence of mitochondrial hypometabolism in both hemispheres. Moreover, metabolism via PPP was reduced bilaterally. Ipsilateral glucose metabolism via PC was reduced, but PC activity was relatively preserved compared with glucose metabolism via pyruvate dehydrogenase. The observed reduction in PPP activity after HI may contribute to the increased susceptibility of the neonatal brain to oxidative stress.     

Outcomes of ceftriaxone use compared to standard of therapy in methicillin susceptible staphylococcal aureus (MSSA) bloodstream infections

Background Standard of care therapy (SOCT) for the treatment of methicillin susceptible staphylococcal aureus (MSSA) infections requires multiple daily infusions. Despite questionable efficacy due to high protein binding, ceftriaxone (CTX) is frequently used for treatment of MSSA at Hines VA Hospital. Objective The objective of this study was to determine clinical and microbiological outcomes in patients with MSSA bacteremia treated with CTX compared to SOCT. Setting This retrospective study was conducted at the Edward Hines, Jr. VA Hospital which is a comprehensive health care center serving the veteran population of the greater metropolitan Chicago and northwest Indiana regions and is institutionally affiliated with the Loyola University Medical Center. The Hines VA provides medical care to over 56,000 veterans and operates approximately 500 hospital beds, including acute care and nursing home beds. Method We conducted a retrospective cohort study of patients with MSSA bacteremia treated at Hines VA Hospital between January 2000 and September 2009. Patients who received either SOCT or CTX for >50 % of the treatment course and for the appropriate duration were included. Patients who were on multiple antibiotics concurrently or who received <14 days of therapy were excluded. Main outcome measure The primary outcome of this study is to compare clinical outcomes of patients with MSSA bacteremia who were treated with CTX compared to those who received standard of care agents. Results Ninety-three patients with MSSA bacteremia were included in the analysis. Fifty-one were treated with SOCT and 42 with CTX. There were no differences in microbiological cure between SOCT (94.1 %) and CTX (95.2 %) (p = 0.812). Clinical cure was similar between groups (74.5 % for SOCT, 83.3 % for CTX) (p = 0.303). CTX was used more often to treat Staphylococcus aureus bacteremia associated with osteomyelitis whereas endocarditis and central line associated infections were treated more frequently with SOCT (p = 0.01). More patients treated with CTX were managed in the ambulatory setting (64 vs. 24 %; p = <0.001). There was a trend toward a longer hospital stay with SOCT. Conclusion Clinical outcomes for MSSA bacteremia did not differ significantly between patients treated with CTX and SOCT. Findings suggest that CTX may be an alternative for outpatient management of MSSA bacteremia.     

Procoagulant soluble tissue factor is released from endothelial cells in response to inflammatory cytokines

Inflammatory cytokines alter the hemostatic balance of endothelial cells (ECs). Alternatively spliced human tissue factor (asHTF), a soluble isoform of tissue factor (TF), has recently been detected in ECs, possibly contributing to procoagulability. Agonists regulating asHTF expression and release are yet unknown. This study examines the effect of TNF-alpha and IL-6 on the endothelial expression of both TF variants and delineates the impact of asHTF on the procoagulability of extracellular fluids. asHTF and TF mRNA were assessed by real-time PCR, and asHTF, TF, and tissue factor pathway inhibitor (TFPI) proteins by Western blot and fluorescence microscopy before and after stimulation with TNF-alpha (10 ng/mL) or IL-6 (10 ng/L). The procoagulability of cell supernatant was analyzed by a chromogenic assay with or without phospholipid vesicles. We found asHTF mRNA to be maximally increased 10 minutes after TNF-alpha and 40 minutes after IL-6 treatment (asHTF/GAPDH ratio 0.0223+/-0.0069 versus 0.0012+/-0.0006 for control, P<0.001 and 0.0022+/-0.0004 versus 0.0012+/-0.0007, P<0.05, respectively). Not only was asHTF increased, but also TFPI decreased after cytokine treatment. asHTF was found in the supernatant as early as 5 hours after TNF-alpha stimulation, supporting factor Xa generation after relipidation (6.55+/-1.13 U versus 2.99+/-0.59 U in control supernatant, P<0.00001). Removal of asHTF from supernatants by immunoprecipitation diminished its procoagulability to baseline. The soluble TF isoform expressed and released from ECs in response to inflammatory cytokines becomes procoagulant in the presence of phospholipids. Thus, asHTF released from ECs is a marker for and a contributor to imbalanced hemostasis.     

Viral myocarditis and coagulopathy: increased tissue factor expression and plasma thrombogenicity

We investigated the effects of viral infection on Tissue Factor (TF) expression and activity in mice within the myocardium to understand increased thrombosis during myocarditis. Mice were infected with coxsackie virus B3 (CVB3) and the hearts were collected at day 4, 8 and 28 post infection (p.i.). Myocardial TF expression and cellular activity as well as plasma activity were analyzed from CVB3 infected mice by Western blot, chromogenic Factor Xa generation assay, in situ staining for active TF and immunohistochemistry. In addition to TF expression, hemodynamic parameters were measured during the time course of infection. Furthermore, we analyzed myocardial tissues from patients with suspected inflammatory cardiomyopathy. TF protein expression was maximally 5-fold elevated 8 days p.i. in mice and remained increased on day 28 p.i. (P < 0.001 vs. non-infected controls). Alterations in TF expression were associated with fibrin deposits within the myocardium. The TF pathway inhibitor protein expression in the myocardium was not altered during myocarditis. Active cellular TF co-localized with CD3 positive cells and VCAM-1 positive endothelial cells in the myocardium. The TF expression was positively correlated with the amount of infiltrating CD3 and Mac3 positive cells (Spearman-Rho ρ = 0.749 P < 0.0001 for CD3⁺ and ρ = 0.775 P < 0.0001 for Mac3⁺; N = 35). Increased myocardial TF expression was associated with a 2-fold elevated plasma activity (P < 0.05 vs. non-infected controls). In the human hearts, the TF expression correlated postively with an endothelial cell activation marker (ρ = 0.523 P < 0.0001 for CD62E; N = 54). Viral myocarditis is a hypercoagulative state which is associated with increased myocardial TF expression and activity. Upregulation of TF contributes to a systemic activation of the coagulation cascade.     

Calcium lability of cytoplasmic microtubules and its modulation by microtubule-associated proteins

Detergent-extracted BSC-1 monkey cells have been used as a model system to study the Ca²⁺ sensitivity of in vivo polymerized microtubules under in vitro conditions. The effects of various experimental treatments were observed by immunofluorescence microscopy. Whereas microtubules are completely stable at Ca²⁺ concentrations below 1 μM, Ca²⁺ at greater than 1-4 μM induces microtubule disassembly that begins in the cell periphery and proceeds towards the cell center. At concentrations of up to 500 μM, both the pattern and time course of disassembly are not markedly altered, suggesting that, within this concentration range, Ca²⁺ effects are catalytic rather than stoichiometric. Higher (millimolar) Ca²⁺ concentration results in rapid destruction of microtubules. Of other divalent cations, only Sr²⁺ has a slight depolymerizing effect, whereas millimolar Ba²⁺, Mg²⁺, or Mn²⁺ is ineffective. Disassembly induced by micromolar Ca²⁺ is inhibited by pharmacological agents known to bind to calmodulin and inhibit its function, suggesting that calmodulin mediates Ca²⁺ effects. Both the addition of exogenous brain microtubule-associated proteins (MAPs) after lysis and the retention of endogenous cellular MAPs normally extracted during the lysis step stabilize microtubules against the depolymerizing effect of micromolar Ca²⁺. The results indicate that, in this model system, microtubules are sensitive to physiological Ca²⁺ concentrations and that this sensitivity may be conferred by calmodulin associated with the microtubules. MAPs appear to have a modulating effect on microtubular Ca²⁺ sensitivity and thus may function as a discriminating factor in cellular functions performed by calmodulin. It is hypothesized that Ca²⁺-stimulated microtubule disassembly depends on the relative amount of MAPs.