bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of TNF‐alpha blockade on coagulopathy and endothelial cell activation in xenoperfused porcine kidneys
Authors: Ramackers Wolf , Klose Johannes , Tiede Andreas , Werwitzke Sonja , Rataj Dennis , Friedrich Lars , Johanning Kai , Vondran Florian W. R. , Bergmann Sabine , Schuettler Wolfgang , Bockmeyer Clemens Luitpold , Becker Jan Ulrich , Klempnauer Jürgen , Winkler Michael
Published: July 2015

Background

Following pig‐to‐primate kidney transplantation, endothelial cell activation and xenogenic activation of the recipient's coagulation eventually leading to organ dysfunction and microthrombosis can be observed. In this study, we examined the effect of a TNF‐receptor fusion protein (TNF‐RFP) on endothelial cell activation and coagulopathy utilizing an appropriate ex vivo perfusion system.

Methods

Using an ex vivo perfusion circuit based on C1‐Inhibitor (C1‐Inh) and low‐dose heparin administration, we have analyzed consumptive coagulopathy following contact of human blood with porcine endothelium. Porcine kidneys were recovered following in situ cold perfusion with Histidine‐tryptophan‐ketoglutarate (HTK) organ preservation solution and were immediately connected to a perfusion circuit utilizing freshly drawn pooled porcine or human AB blood. The experiments were performed in three individual groups: autologous perfusion (n = 5), xenogenic perfusion without any further pharmacological intervention (n = 10), or with addition of TNF‐RFP (n = 5). After perfusion, tissue samples were obtained for real‐time PCR and immunohistological analyses. Endothelial cell activation was assessed by measuring the expression levels of E‐selectin, ICAM‐1, and VCAM‐1.

Results

Kidney survival during organ perfusion with human blood, C1‐Inh, and heparin, but without any further pharmacological intervention was 126 ± 78 min. Coagulopathy was observed with significantly elevated concentrations of D‐dimer and thrombin–antithrombin complex (TAT), resulting in the formation of multiple microthrombi. Endothelial cell activation was pronounced, as shown by increased expression of E‐selectin and VCAM‐1. In contrast, pharmacological intervention with TNF‐RFP prolonged organ survival to 240 ± 0 min (max. perfusion time; no difference to autologous control). Formation of microthrombi was slightly reduced, although not significantly, if compared to the xenogenic control. D‐dimer and TAT were elevated at similar levels to the xenogenic control experiments. In contrast, endothelial cell activation, as shown by real‐time PCR, was significantly reduced in the TNF‐RFP group.

Conclusion

We conclude that although coagulopathy was not affected, TNF‐RFP is able to suppress inflammation occurring after xenoperfusion in this ex vivo perfusion model.
Restricting unipotent characters in special orthogonal groups
Authors: Himstedt Frank , Noeske Felix
Published: July 2015

For all prime powers $q$ we restrict the unipotent characters of the special orthogonal groups $\text{SO}_{5}(q)$ and $\text{SO}_{7}(q)$ to a maximal parabolic subgroup. We determine all irreducible constituents of these restrictions for $\text{SO}_{5}(q)$ and a large part of the irreducible constituents for $\text{SO}_{7}(q)$.
Design Improvement Survey for NOx Emissions Reduction of a Heavy-Duty Gas Turbine Partially Premixed Fuel Nozzle Operating With Natural Gas: Numerical Assessment
Authors: Innocenti Alessandro , Andreini Antonio , Facchini Bruno , Cerutti Matteo , Ceccherini Gianni , Riccio Giovanni
Published: July 2015

A numerical investigation of a low NOx partially premixed fuel nozzle for heavy-duty gas turbine applications is presented in this paper. Availability of results from a recent test campaign on the same fuel nozzle architecture allowed the exhaustive comparison study presented in this work. At first, an assessment of the turbulent combustion model was carried out, with a critical investigation of the expected turbulent combustion regimes in the system and taking into account the partially premixed nature of the flame due to the presence of diffusion type pilot flames. In particular, the fluent partially premixed combustion model and a flamelet approach are used to simulate the flame. The laminar flamelet database is generated using the flamelet generated manifold (FGM) chemistry reduction technique. Species and temperature are parameterized by mixture fraction and progress variable. Comparisons with calculations with partially premixed model and the steady diffusion flamelet (SDF) database are made for the baseline configuration in order to discuss possible gains associated with the introduced dimension in the FGM database (reaction progress), which makes it possible to account for nonequilibrium effects. Numerical characterization of the baseline nozzle has been carried out in terms of NOx. Computed values for both the baseline and some alternative premixer designs have been then compared with experimental measurements on the reactive test rig at different operating conditions and different split ratios between main and pilot fuel. Numerical results allowed pointing out the fundamental NOx formation processes, both in terms of spatial distribution within the flame and in terms of different formation mechanisms. The obtained knowledge would allow further improvement of fuel nozzle design.
Passivation of oxide traps in gallium arsenide (semiconductor) metal-oxide-semiconductor capacitor with high-k dielectric by using fluorine incorporation
Authors: Liu Lining , Choi Hoi Wai , Lai Pui To , Xu Jingping
Published: July 2015

Gallium arsenide (semiconductor) (GaAs) metal-oxide-semiconductor capacitors with fluorine-incorporated TaHfON as gate dielectric are fabricated by pre- or postdeposition fluorine plasma treatment and their electrical and physical properties are compared with a control sample without the treatment. Among the three devices, the one with postdeposition fluorine treatment exhibits better characteristics: low oxide-charge density (−3.5 × 1012 cm−2), low interface-state density (2.2 × 1012 cm−2eV−1), small flatband voltage (0.7 V), small hysteresis (45 mV), and good capacitance–voltage behavior. These should be attributed to (1) the passivating effects of fluorine atoms on the acceptorlike interface and near-interface traps, and (2) fluorine-induced suppressed growth of unstable Ga and As oxides on the GaAs substrate during postdeposition annealing.
NF-κB signaling relieves negative regulation by miR-194 in hepatocellular carcinoma by suppressing the transcription factor HNF-1α
Authors: Bao Chunyang , Li Yan , Huan Lin , Zhang Yuannv , Zhao Fangyu , Wang Qifeng , Liang Linhui , Ding Jie , Liu Li , Chen Taoyang , Li Jinjun , Yao Ming , Huang Shenglin , He Xianghuo
Published: July 2015

Activation of NF-κB signaling in liver cancer cells suppresses a microRNA that usually limits NF-κB activity.
HDL-S1P limits hematopoiesis
Authors: VanHook Annalisa M.
Published: July 2015

HDL binding is required for S1P to limit lymphopoiesis but not to promote lymphocyte egress.
Allergic sensations fuel asthma
Authors: Ferrarelli Leslie K.
Published: July 2015

Silencing sensory neurons in the lungs using a sodium channel inhibitor may dampen the asthmatic response to allergens.
Preventing photoreceptor death after retinal injury
Authors: Smith Orla
Published: July 2015

Inhibiting the alternative complement pathway prevents injury-induced photoreceptor cell death.
Eicosanoids enhance transplant success
Authors: VanHook Annalisa M.
Published: July 2015

Epoxyeicosatrienoic acids promote hematopoiesis and enhance bone marrow transplant success.
Pancreatic crosstalk among beta and delta cells
Authors: Wong Wei
Published: July 2015

A peptide hormone that is coreleased with insulin triggers somatostatin secretion to ultimately limit insulin secretion.
Population Pharmacokinetics and Target Attainment of Meropenem in Plasma and Tissue of Morbidly Obese Patients after Laparoscopic Intraperitoneal Surgery
Authors: Wittau Mathias , Scheele Jan , Kurlbaum Max , Brockschmidt Claas , Wolf Anna M. , Hemper Evelyn , Henne-Bruns Doris , Bulitta Jürgen B.
Published: July 2015

Meropenem serves as a clinically important, broad-spectrum antibiotic. While meropenem is commonly used in obese patients, its pharmacokinetics in this patient group is not well known. Our aim was to characterize the population pharmacokinetics and target attainment in plasma, subcutaneous tissue, and peritoneal fluid for meropenem in morbidly obese patients. Four doses of 1g meropenem were given as 15-min infusions every 8 h to five morbidly obese patients (body mass index [BMI], 47.6 to 62.3 kg/m
2
). After the fourth dose, serial meropenem concentrations were determined in plasma and, via microdialysis, in subcutaneous tissue and peritoneal fluid. All concentrations were analyzed simultaneously via population modeling, and target attainment probabilities predicted via Monte Carlo simulations using the target of unbound meropenem concentrations above the MIC for at least 40% of the dosing interval. For patients with 53 kg fat-free mass, total clearance was 18.7 liters/h and volume of distribution at steady state was 27.6 liters. The concentrations in subcutaneous tissue and peritoneal fluid largely paralleled those in plasma (equilibration half-life, <30 min). The area under the curve (AUC) in subcutaneous tissue divided by the plasma AUC had a mean of 0.721. For peritoneal fluid, this AUC ratio had a mean of 0.943. Target attainment probabilities were >90% after 1 g meropenem every 8 h as a 15-min infusion for MICs of up to 2 mg/liter in plasma and peritoneal fluid and 0.5 mg/liter in subcutaneous tissue. Meropenem pharmacokinetics in plasma and peritoneal fluid of obese patients was predictable, but subcutaneous tissue penetration varied greatly. (This study has been registered at ClinicalTrials.gov under registration no. NCT01407965.)
In Vitro
Activities of Tedizolid and Linezolid against Gram-Positive Cocci Associated with Acute Bacterial Skin and Skin Structure Infections and Pneumonia
Authors: Chen Ko-Hung , Huang Yu-Tsung , Liao Chun-Hsing , Sheng Wang-Hui , Hsueh Po-Ren
Published: July 2015

Tedizolid is a novel, expanded-spectrum oxazolidinone with potent activity against a wide range of Gram-positive pathogens. A total of 425 isolates of Gram-positive bacteria were obtained consecutively from patients with acute bacterial skin and skin structure infections (ABSSSIs) or pneumonia. These isolates included methicillin-susceptible
Staphylococcus aureus
(MSSA) (
n
= 100), methicillin-resistant
Staphylococcus aureus
(MRSA) (
n
= 100),
Streptococcus pyogenes
(
n
= 50),
Streptococcus agalactiae
(
n
= 50),
Streptococcus anginosus
group (
n
= 75),
Enterococcus faecalis
(
n
= 50), and vancomycin-resistant enterococci (VRE) (
Enterococcus faecium
) (
n
= 50). The MICs of tedizolid and linezolid were determined by the agar dilution method. Tedizolid exhibited better
in vitro
activities than linezolid against MSSA (MIC
90
s, 0.5 versus 2 μg/ml), MRSA (MIC
90
s, 0.5 versus 2 μg/ml),
S. pyogenes
(MIC
90
s, 0.5 versus 2 μg/ml),
S. agalactiae
(MIC
90
s, 0.5 versus 2 μg/ml),
Streptococcus anginosus
group (MIC
90
s, 0.5 versus 2 μg/ml),
E. faecalis
(MIC
90
s, 0.5 versus 2 μg/ml), and VRE (MIC
90
s, 0.5 versus 2 μg/ml). The tedizolid MICs against
E. faecalis
(
n
= 3) and VRE (
n
= 2) intermediate to linezolid (MICs, 4 μg/ml) were 1 μg/ml and 0.5 μg/ml, respectively. The tedizolid MIC
90
s against S
. anginosus
,
S. constellatus
, and
S. intermedius
were 0.5, 1, and 0.5 μg/ml, respectively, and the rates of susceptibility based on the U.S. FDA MIC interpretive breakpoints to the isolates were 16%, 28%, and 72%, respectively. Tedizolid exhibited 2- to 4-fold better
in vitro
activities than linezolid against a variety of Gram-positive cocci associated with ABSSSIs and pneumonia. The lower susceptibilities of tedizolid against isolates of
S. anginosus
and
S. constellatus
than against those of
S. intermedius
in Taiwan were noted.
Multicenter, Double-Blind, Randomized, Phase 2 Study Evaluating the Novel Antibiotic Cadazolid in Patients with Clostridium difficile Infection
Authors: Louie Thomas , Nord Carl Erik , Talbot George H. , Wilcox Mark , Gerding Dale N. , Buitrago Martha , Kracker Hilke , Charef Pascal , Cornely Oliver A.
Published: July 2015

Cadazolid, a novel fluoroquinolone-oxazolidinone antibiotic, exhibits potent
in vitro
activity against
Clostridium difficile
, including the epidemic BI/NAP1/027 strain. This multicenter, randomized, double-blind, active reference group, phase 2 study evaluated the efficacy and safety of oral cadazolid in treatment of adult patients with
C. difficile
infection (CDI). Eligible patients with first occurrence/first recurrence of CDI were randomized 1:1:1:1 to 250, 500, or 1,000 mg cadazolid twice daily (BID) or oral 125 mg vancomycin four times daily (QID) for 10 days. The primary endpoint was clinical cure at test of cure (48 ± 24 h after the end of treatment; modified intent-to-treat population), defined as resolution of diarrhea with no further CDI treatment required. Secondary endpoints included recurrence rate, sustained clinical response (clinical cure without recurrence), and time to diarrhea resolution. Of 84 patients enrolled, 20, 22, 20, and 22 received 250, 500, or 1,000 mg cadazolid BID or 125 mg vancomycin QID, respectively. The primary endpoint was achieved in 76.5% (80% confidence interval [CI], 58.4, 89.3), 80.0% (63.9, 91.0), 68.4% (51.1, 82.5), and 68.2% (52.3, 81.3) of patients, respectively. There was no evidence of a cadazolid dosage-dependent response. Each dosage of cadazolid resulted in a lower recurrence rate than with vancomycin (18.2 to 25.0% versus 50%). Consequently, higher sustained clinical response rates were observed with cadazolid (46.7 to 60.0%) than with vancomycin (33.3%). The times to diarrhea resolution were similar for cadazolid and vancomycin. Cadazolid was well tolerated, with no safety signal observed. The results of this phase 2 study support further clinical development of cadazolid. (This study has been registered in the United States at ClinicalTrials.gov under registration no. NCT01222702 and in Europe with the European Medicines Agency under registration no. EUDRA-CT 2010-020941-29.)
New Insights Regarding Yeast Survival following Exposure to Liposomal Amphotericin B
Authors: Teixeira-Santos Rita , Ricardo Elisabete , Guerreiro Susana Gomes , Costa-de-Oliveira Sofia , Rodrigues Acácio Gonçalves , Pina-Vaz Cidália
Published: July 2015

In vitro
resistance to amphotericin B is an extremely rare event among pathogenic yeasts. However,
in vivo
response is sometimes reduced, resulting in an unfavorable outcome. Such adverse outcomes might be related to subfungicidal plasma concentrations. We aimed to clarify the mechanisms of liposomal amphotericin B (AMB-L; AmBisome)-induced lesions and the mechanisms responsible for yeast cell recovery following exposure at plasma concentrations. The physiological statuses developing following exposure to AMB-L at simulated plasma concentrations (20 to 0.1 mg/liter) and at a constant concentration (3 mg/liter) were assessed in a 24-h time course assay. Time-kill experiments also were carried out under the same AMB-L treatment conditions. Our results suggest that yeast cells develop compensatory responses related to membrane polarization, metabolic activity, and reactive oxygen species (ROS) production after exposure to high plasma concentrations (20 to 5 mg/liter) during the first 6 h; in the remaining 18 h, when exposed to lower concentrations, cells reveal almost full recovery with no evidence of fungicidal activity. In contrast, whenever cells are exposed to a constant concentration above the MIC, despite initially exhibiting compensatory stress responses, soon afterwards they exhibit membrane depolarization, a decrease of metabolic activity, increasing ROS production, and lastly, programmed cell death and necrosis, resulting in succumbing to AMB-L fungicidal effects. This study may represent a step forward in the support of AMB-L use for clinical treatment of invasive fungal infections, since it demonstrates the importance of maintaining levels of AMB-L above the MIC in plasma and tissues to ensure it produces its fungicidal effects.
Exposure-Response Analyses for Tafenoquine after Administration to Patients with Plasmodium vivax Malaria
Authors: Tenero David , Green Justin A. , Goyal Navin
Published: July 2015

Tafenoquine (TQ), a new 8-aminoquinoline with activity against all stages of the
Plasmodium vivax
life cycle, is being developed for the radical cure of acute
P. vivax
malaria in combination with chloroquine. The efficacy and exposure data from a pivotal phase 2b dose-ranging study were used to conduct exposure-response analyses for TQ after administration to subjects with
P. vivax
malaria. TQ exposure (i.e., area under the concentration-time curve [AUC]) and region (Thailand compared to Peru and Brazil) were found to be statistically significant predictors of clinical response based on multivariate logistic regression analyses. After accounting for region/country, the odds of being relapse free at 6 months increased by approximately 51% (95% confidence intervals [CI], 25%, 82%) for each 25-U increase in AUC above the median value of 54.5 μg · h/ml. TQ exposure was also a significant predictor of the time to relapse of the infection. The final parametric, time-to-event model for the time to relapse, included a Weibull distribution hazard function, AUC, and country as covariates. Based on the model, the risk of relapse decreased by 30% (95% CI, 17% to 42%) for every 25-U increase in AUC. Monte Carlo simulations indicated that the 300-mg dose of TQ would provide an AUC greater than the clinically relevant breakpoint obtained in a classification and regression tree (CART) analysis (56.4 μg · h/ml) in more than 90% of subjects and consequently result in a high probability of being relapse free at 6 months. This model-based approach was critical in selecting an appropriate phase 3 dose. (This study has been registered at ClinicalTrials.gov under registration no. NCT01376167.)
Racial Differences in Clostridium difficile Infection Rates Are Attributable to Disparities in Health Care Access
Authors: Mao Eric J. , Kelly Colleen R. , Machan Jason T.
Published: July 2015

This study confirms previously reported racial differences in
Clostridium difficile
infection (CDI) rates in the United States and explores the nature of those differences. We conducted a retrospective study using the 2010 Nationwide Inpatient Sample, the largest all-payer database of hospital discharges in the United States. We identified hospital stays most likely to include antibiotic treatment for infections, based on hospital discharge diagnoses, and we examined how CDI rates varied, in an attempt to distinguish between genotypic and environmental racial differences. Logistic regressions for the survey design were used to test hypotheses. Among patients likely to have received antibiotics, white patients had higher CDI rates than black, Hispanic, Asian, and Native American patients (
P
< 0.0001). CDI rates increased with higher income levels and were higher for hospitalizations paid by private insurance versus those paid by Medicaid or classified as self-pay or free care (
P
< 0.0001). Among patients admitted from skilled nursing facilities, where racial bias in health care access is less, racial differences in CDI rates disappeared (
P
= 1.0). Infected patients did not show racial differences in rates of complicated CDI or death (
P
= 1.0). Although white patients had greater CDI rates than nonwhite patients, racial differences in CDI rates disappeared in a population for which health care access was presumed to be less racially biased. This provides evidence that apparent racial differences in CDI risks may represent health care access disparities, rather than genotypic differences. CDI represents a deviation from the paradigm that increased health care access is associated with less morbidity.
Mutations in β-Lactamase AmpC Increase Resistance of Pseudomonas aeruginosa Isolates to Antipseudomonal Cephalosporins
Authors: Berrazeg M. , Jeannot K. , Ntsogo Enguéné Véronique Yvette , Broutin I. , Loeffert S. , Fournier D. , Plésiat P.
Published: July 2015

Mutation-dependent overproduction of intrinsic β-lactamase AmpC is considered the main cause of resistance of clinical strains of
Pseudomonas aeruginosa
to antipseudomonal penicillins and cephalosporins. Analysis of 31 AmpC-overproducing clinical isolates exhibiting a greater resistance to ceftazidime than to piperacillin-tazobactam revealed the presence of 17 mutations in the β-lactamase, combined with various polymorphic amino acid substitutions. When overexpressed in AmpC-deficient
P. aeruginosa
4098, the genes coding for 20/23 of these AmpC variants were found to confer a higher (2-fold to >64-fold) resistance to ceftazidime and ceftolozane-tazobactam than did the gene from reference strain PAO1. The mutations had variable effects on the MICs of ticarcillin, piperacillin-tazobactam, aztreonam, and cefepime. Depending on their location in the AmpC structure and their impact on β-lactam MICs, they could be assigned to 4 distinct groups. Most of the mutations affecting the omega loop, the R2 domain, and the C-terminal end of the protein were shared with extended-spectrum AmpCs (ESACs) from other Gram-negative species. Interestingly, two new mutations (F121L and P154L) were predicted to enlarge the substrate binding pocket by disrupting the stacking between residues F121 and P154. We also found that the reported ESACs emerged locally in a variety of clones, some of which are epidemic and did not require hypermutability. Taken together, our results show that
P. aeruginosa
is able to adapt to efficacious β-lactams, including the newer cephalosporin ceftolozane, through a variety of mutations affecting its intrinsic β-lactamase, AmpC. Data suggest that the rates of ESAC-producing mutants are ≥1.5% in the clinical setting.
The Glucose Transporter PfHT1 Is an Antimalarial Target of the HIV Protease Inhibitor Lopinavir
Authors: Kraft Thomas E. , Armstrong Christopher , Heitmeier Monique R. , Odom Audrey R. , Hruz Paul W.
Published: July 2015

Malaria and HIV infection are coendemic in a large portion of the world and remain a major cause of morbidity and mortality. Growing resistance of
Plasmodium
species to existing therapies has increased the need for new therapeutic approaches. The
Plasmodium
glucose transporter PfHT is known to be essential for parasite growth and survival. We have previously shown that HIV protease inhibitors (PIs) act as antagonists of mammalian glucose transporters. While the PI lopinavir is known to have antimalarial activity, the mechanism of action is unknown. We report here that lopinavir blocks glucose uptake into isolated malaria parasites at therapeutically relevant drug levels. Malaria parasites depend on a constant supply of glucose as their primary source of energy, and decreasing the available concentration of glucose leads to parasite death. We identified the malarial glucose transporter PfHT as a target for inhibition by lopinavir that leads to parasite death. This discovery provides a mechanistic basis for the antimalarial effect of lopinavir and provides a direct target for novel drug design with utility beyond the HIV-infected population.
Role of Interleukin-12 in Protection against Pulmonary Infection with Methicillin-Resistant Staphylococcus aureus
Authors: Nguyen Quang-Tam , Furuya Yoichi , Roberts Sean , Metzger Dennis W.
Published: July 2015

Methicillin-resistantStaphylococcus aureus(MRSA) is a common pathogen associated with nosocomial pneumonia and is an increasing threat for severe community-acquired pneumonia. We have now investigated the role of interleukin-12 (IL-12) in protective immunity against lung infection with MRSA. The importance of IL-12 in protection from pulmonary MRSA infection was demonstrated by the finding that IL-12p35-deficient mice had a lower survival rate, higher bacterial burdens in lung and spleen, and decreased expression of interferon gamma (IFN-γ) in the lung compared to wild-type mice. These effects were completely reversed by replacement intranasal therapy with recombinant IL-12. Furthermore, exogenous IL-12 treatment of wild-type mice 24 h before pulmonary challenge with a lethal dose of MRSA significantly improved bacterial clearance and resulted in protection from death. The IL-12-treated mice had increased numbers of lung natural killer (NK) cells and neutrophils and higher levels of IFN-γ in the lung and serum compared to untreated mice. The major source of IL-12-driven IFN-γ expression in the lung was the NK cell, and the direct target of pulmonary IFN-γ was the lung macrophage, as shown using mice with a macrophage-specific defect in interferon gamma (IFN-γ) signaling (MIIG mice). Importantly, combination therapy with linezolid and IL-12 following intranasal MRSA infection significantly increased survival compared to that of mice receiving linezolid or IL-12 alone. These results indicate that IL-12-based immunotherapy may hold promise for treatment of MRSA pneumonia.
Modulation of
ccrAB
Expression and SCC
mec
Excision by an Inverted Repeat Element and SarS in Methicillin-Resistant Staphylococcus aureus
Authors: Zhang Shijie , Ma Ronghua , Liu Xiaoyu , Zhang Xu , Sun Baolin
Published: July 2015

Methicillin-resistant
Staphylococcus aureus
(MRSA) is a notorious human pathogen that can cause a broad spectrum of infections. MRSA strains are resistant to almost the entire family of β-lactam antibiotics due to the acquisition of staphylococcal cassette chromosome
mec
(SCC
mec
). The chromosome cassette recombinases A and B, encoded by
ccrAB
genes located on SCC
mec
, play a key role in the excision of SCC
mec
. Studies have shown that
ccrAB
genes are expressed in only a minority of cells, suggesting the involvement of a subtle regulatory mechanism in
ccrAB
expression which has not been uncovered. Here, we found that an inverted repeat (IR) element, existing extensively and conservatively within the
ccrAB
promoter of different SCC
mec
types, played a repressive role in
ccrAB
expression and SCC
mec
excision in MRSA strain N315. Replacement of the IR sequence led to a significant increase in
ccrAB
expression and curing of SCC
mec
from strain N315 cells. In addition, we identified the transcriptional regulator SarS using DNA-affinity chromatography and further demonstrated that SarS can bind to the IR sequence and upregulate
ccrAB
expression and SCC
mec
excision. These findings reveal a molecular mechanism regulating
ccrAB
expression and SCC
mec
excision and may provide mechanic insights into the lateral transfer of SCC
mec
and spread of antibiotic resistance in
S. aureus
.
Amikacin Concentrations Predictive of Ototoxicity in Multidrug-Resistant Tuberculosis Patients
Authors: Modongo Chawangwa , Pasipanodya Jotam G. , Zetola Nicola M. , Williams Scott M. , Sirugo Giorgio , Gumbo Tawanda
Published: July 2015

Aminoglycosides, such as amikacin, are used to treat multidrug-resistant tuberculosis. However, ototoxicity is a common problem and is monitored using peak and trough amikacin concentrations based on World Health Organization recommendations. Our objective was to identify clinical factors predictive of ototoxicity using an agnostic machine learning method. We used classification and regression tree (CART) analyses to identify clinical factors, including amikacin concentration thresholds that predicted audiometry-confirmed ototoxicity among 28 multidrug-resistant pulmonary tuberculosis patients in Botswana. Amikacin concentrations were measured for all patients. The quantitative relationship between predictive factors and the probability of ototoxicity were then identified using probit analyses. The primary predictors of ototoxicity on CART analyses were cumulative days of therapy, followed by cumulative area under the concentration-time curve (AUC), which improved on the primary predictor by 87%. The area under the receiver operating curve was 0.97 on the test set. Peak and trough were not predictors in any tree. When algorithms were forced to pick peak and trough as primary predictors, the area under the receiver operating curve fell to 0.46. Probit analysis revealed that the probability of ototoxicity increased sharply starting after 6 months of therapy to near maximum at 9 months. A 10% probability of ototoxicity occurred with a threshold cumulative AUC of 87,232 days · mg · h/liter, while that of 20% occurred at 120,000 days · mg · h/liter. Thus, cumulative amikacin AUC and duration of therapy, and not peak and trough concentrations, should be used as the primary decision-making parameters to minimize the likelihood of ototoxicity in multidrug-resistant tuberculosis.
Elevated Expression of GlpT and UhpT via FNR Activation Contributes to Increased Fosfomycin Susceptibility in Escherichia coli under Anaerobic Conditions
Authors: Kurabayashi Kumiko , Tanimoto Koichi , Fueki Shinobu , Tomita Haruyoshi , Hirakawa Hidetada
Published: July 2015

Because a shortage of new antimicrobial agents is a critical issue at present, and with the spread of multidrug-resistant (MDR) pathogens, the use of fosfomycin to treat infections is being revisited as a “last-resort option.” This drug offers a particular benefit in that it is more effective against bacteria growing under oxygen-limited conditions, unlike other commonly used antimicrobials, such as fluoroquinolones and aminoglycosides. In this study, we showed that
Escherichia coli
strains, including enterohemorrhagic
E. coli
(EHEC), were more susceptible to fosfomycin when grown anaerobically than when grown aerobically, and we investigated how the activity of this drug was enhanced during anaerobic growth of
E. coli
. Our quantitative PCR analysis and a transport assay showed that
E. coli
cells grown under anaerobic conditions had higher levels of expression of
glpT
and
uhpT
, encoding proteins that transport fosfomycin into cells with their native substrates, i.e., glycerol-3-phosphate and glucose-6-phosphate, and led to increased intracellular accumulation of the drug. Elevation of expression of these genes during anaerobic growth requires FNR, a global transcriptional regulator that is activated under anaerobic conditions. Purified FNR bound to DNA fragments from regions upstream of
glpT
and
uhpT
, suggesting that it is an activator of expression of
glpT
and
uhpT
during anaerobic growth. We concluded that the increased antibacterial activity of fosfomycin toward
E. coli
under anaerobic conditions can be attributed to elevated expression of GlpT and UhpT following activation of FNR, leading to increased uptake of the drug.
Key Residues of Outer Membrane Protein OprI Involved in Hexamer Formation and Bacterial Susceptibility to Cationic Antimicrobial Peptides
Authors: Chang Ting-Wei , Wang Chiu-Feng , Huang Hsin-Jye , Wang Iren , Hsu Shang-Te Danny , Liao You-Di
Published: July 2015

Antimicrobial peptides (AMPs) are important components of the host innate defense mechanism against invading pathogens. Our previous studies have shown that the outer membrane protein, OprI from
Pseudomonas aeruginosa
or its homologue, plays a vital role in the susceptibility of Gram-negative bacteria to cationic α-helical AMPs (Y. M. Lin, S. J. Wu, T. W. Chang, C. F. Wang, C. S. Suen, M. J. Hwang, M. D. Chang, Y. T. Chen, Y. D. Liao, J Biol Chem 285:8985–8994, 2010,
http://dx.doi.org/10.1074/jbc.M109.078725
; T. W. Chang, Y. M. Lin, C. F. Wang, Y. D. Liao, J Biol Chem
287:
418–428, 2012,
http://dx.doi.org/10.1074/jbc.M111.290361
). Here, we obtained two forms of recombinant OprI: rOprI-F, a hexamer composed of three disulfide-bridged dimers, was active in AMP binding, while rOprI-R, a trimer, was not. All the subunits predominantly consisted of α-helices and exhibited rigid structures with a melting point centered around 76°C. Interestingly, OprI tagged with
Escherichia coli
signal peptide was expressed in a hexamer, which was anchored on the surface of
E. coli
, possibly through lipid acids added at the N terminus of OprI and involved in the binding and susceptibility to AMP as native
P. aeruginosa
OprI. Deletion and mutation studies showed that Cys1 and Asp27 played a key role in hexamer formation and AMP binding, respectively. The increase of OprI hydrophobicity upon AMP binding revealed that it undergoes conformational changes for membrane fusion. Our results showed that OprI on bacterial surfaces is responsible for the recruitment and susceptibility to amphipathic α-helical AMPs and may be used to screen antimicrobials.
Antimicrobial Susceptibility Profiles of Staphylococcus aureus Isolates Recovered from Humans, Environmental Surfaces, and Companion Animals in Households of Children with Community-Onset Methicillin-Resistant S. aureus Infections
Authors: Morelli John J. , Hogan Patrick G. , Sullivan Melanie L. , Muenks Carol E. , Wang Jeffrey W. , Thompson Ryley M. , Burnham Carey-Ann D. , Fritz Stephanie A.
Published: July 2015

Our objective was to determine the antibiotic susceptibility profiles of
Staphylococcus aureus
isolates recovered from 110 households of children with community-onset methicillin-resistant
S. aureus
(MRSA) infections. Cultures were obtained from household members, household objects, and dogs and cats, yielding 1,633
S. aureus
isolates. The
S. aureus
isolates were heterogeneous, although more than half were methicillin resistant. The highest proportion of MRSA was found in bathrooms. The majority of isolates were susceptible to antibiotics prescribed in outpatient settings.
Intestinal Microbiota of Mice Influences Resistance to Staphylococcus aureus Pneumonia
Authors: Gauguet Stefanie , D'Ortona Samantha , Ahnger-Pier Kathryn , Duan Biyan , Surana Neeraj K. , Lu Roger , Cywes-Bentley Colette , Gadjeva Mihaela , Shan Qiang , Priebe Gregory P. , Pier Gerald B.
Published: July 2015

Th17 immunity in the gastrointestinal tract is regulated by the intestinal microbiota composition, particularly the presence of segmented filamentous bacteria (sfb), but the role of the intestinal microbiota in pulmonary host defense is not well explored. We tested whether altering the gut microbiota by acquiring sfb influences the susceptibility to staphylococcal pneumonia via induction of type 17 immunity. Groups of C57BL/6 mice which differed in their intestinal colonization with sfb were challenged with methicillin-resistant
Staphylococcus aureus
in an acute lung infection model. Bacterial burdens, bronchoalveolar lavage fluid (BALF) cell counts, cell types, and cytokine levels were compared between mice from different vendors, mice from both vendors after cohousing, mice given sfb orally prior to infection, and mice with and without exogenous interleukin-22 (IL-22) or anti-IL-22 antibodies. Mice lacking sfb developed more severe
S. aureus
pneumonia than mice colonized with sfb, as indicated by higher bacterial burdens in the lungs, lung inflammation, and mortality. This difference was reduced when sfb-negative mice acquired sfb in their gut microbiota through cohousing with sfb-positive mice or when given sfb orally. Levels of type 17 immune effectors in the lung were higher after infection in sfb-positive mice and increased in sfb-negative mice after acquisition of sfb, as demonstrated by higher levels of IL-22 and larger numbers of IL-22
+
TCRβ
+
cells and neutrophils in BALF. Exogenous IL-22 protected mice from
S. aureus
pneumonia. The murine gut microbiota, particularly the presence of sfb, promotes pulmonary type 17 immunity and resistance to
S. aureus
pneumonia, and IL-22 protects against severe pulmonary staphylococcal infection.
Influenza and Bacterial Superinfection: Illuminating the Immunologic Mechanisms of Disease
Authors: Rynda-Apple Agnieszka , Robinson Keven M. , Alcorn John F.
Published: July 2015

Seasonal influenza virus infection presents a major strain on the health care system. Influenza virus infection has pandemic potential, which was repeatedly observed during the last century. Severe disease may occur in the young, in the elderly, in those with preexisting lung disease, and in previously healthy individuals. A common cause of severe influenza pathogenesis is superinfection with bacterial pathogens, namely,
Staphylococcus aureus
and
Streptococcus pneumoniae
. A great deal of recent research has focused on the immune pathways involved in influenza-induced susceptibility to secondary bacterial pneumonia. Both innate and adaptive antibacterial host defenses are impaired in the context of preceding influenza virus infection. The goal of this minireview is to highlight these findings and synthesize these data into a shared central theme of pathogenesis.
Multiple Plasmodium falciparum Erythrocyte Membrane Protein 1 Variants per Genome Can Bind IgM via Its Fc Fragment Fcμ
Authors: Jeppesen Anine , Ditlev Sisse Bolm , Soroka Vladyslav , Stevenson Liz , Turner Louise , Dzikowski Ron , Hviid Lars , Barfod Lea
Published: July 2015

ThePlasmodium falciparumerythrocyte membrane protein 1 (PfEMP1) adhesive proteins expressed on the surfaces of infected erythrocytes (IEs) are of key importance in the pathogenesis ofP. falciparummalaria. Several structurally and functionally defined PfEMP1 types have been associated with severe clinical manifestations, such as cerebral malaria in children and placental malaria in pregnant women. PfEMP1 that can bind the Fc part of IgM (Fcμ) characterizes one such type, although the functional significance of this IgM binding to PfEMP1 remains unclear. In this study, we report the identification and functional analysis of five IgM-binding PfEMP1 proteins encoded byP. falciparumNF54. In addition to the VAR2CSA-type PFL0030c protein, already known to bind Fcμ and to mediate chondroitin sulfate A (CSA)-specific adhesion of IEs in the placenta, we found four PfEMP1 proteins not previously known to bind IgM this way. Although they all contained Duffy binding-like ε (DBLε) domains similar to those in VAR2CSA-type PfEMP1, they did not mediate IE adhesion to CSA, and IgM binding did not shield IEs from phagocytosis of IgG-opsonized IEs. In this way, these new IgM-binding PfEMP1 proteins resemble the rosette-mediating and IgM-binding PfEMP1 HB3VAR06, but none of them mediated formation of rosettes. We could map the capacity for Fc-specific IgM binding to DBLε domains near the C terminus for three of the four PfEMP1 proteins tested. Our study provides new evidence regarding Fc-dependent binding of IgM to PfEMP1, which appears to be a common and multifunctional phenotype.
Dot/Icm Effector Translocation by Legionella longbeachae Creates a Replicative Vacuole Similar to That of Legionella pneumophila despite Translocation of Distinct Effector Repertoires
Authors: Wood Rebecca E. , Newton Patrice , Latomanski Eleanor A. , Newton Hayley J.
Published: July 2015

Legionella
organisms are environmental bacteria and accidental human pathogens that can cause severe pneumonia, termed Legionnaires' disease. These bacteria replicate within a pathogen-derived vacuole termed the
Legionella
-containing vacuole (LCV). Our understanding of the development and dynamics of this vacuole is based on extensive analysis of
Legionella pneumophila
. Here, we have characterized the
Legionella longbeachae
replicative vacuole (
longbeachae
-LCV) and demonstrated that, despite important genomic differences, key features of the replicative LCV are comparable to those of the LCV of
L. pneumophila
(
pneumophila
-LCV). We constructed a Dot/Icm-deficient strain by deleting
dotB
and demonstrated the inability of this mutant to replicate inside THP-1 cells.
L. longbeachae
does not enter THP-1 cells as efficiently as
L. pneumophila
, and this is reflected in the observation that translocation of BlaM-RalF
LLO
(where RalF
LLO
is the
L. longbeachae
homologue of RalF) into THP-1 cells by the
L. longbeachae
Dot/Icm system is less efficient than that by
L. pneumophila
. This difference is negated in A549 cells where
L. longbeachae
and
L. pneumophila
infect with similar entry dynamics. A β-lactamase assay was employed to demonstrate the translocation of a novel family of proteins, the
R
ab-
l
ike
e
ffector (Rle) proteins. Immunofluorescence analysis confirmed that these proteins enter the host cell during infection and display distinct subcellular localizations, with RleA and RleC present on the
longbeachae
-LCV. We observed that the host Rab GTPase, Rab1, and the v-SNARE Sec22b are also recruited to the
longbeachae
-LCV during the early stages of infection, coinciding with the LCV avoiding endocytic maturation. These studies further our understanding of the
L. longbeachae
replicative vacuole, highlighting phenotypic similarities to the vacuole of
L. pneumophila
as well as unique aspects of LCV biology.
Activation of Hepatic STAT3 Maintains Pulmonary Defense during Endotoxemia
Authors: Hilliard Kristie L. , Allen Eri , Traber Katrina E. , Kim Yuri , Wasserman Gregory A. , Jones Matthew R. , Mizgerd Joseph P. , Quinton Lee J.
Published: July 2015

Pneumonia and infection-induced sepsis are worldwide public health concerns. Both pathologies elicit systemic inflammation and induce a robust acute-phase response (APR). Although APR activation is well regarded as a hallmark of infection, the direct contributions of liver activation to pulmonary defense during sepsis remain unclear. By targeting STAT3-dependent acute-phase changes in the liver, we evaluated the role of liver STAT3 activity in promoting host defense in the context of sepsis and pneumonia. We employed a two-hit endotoxemia/pneumonia model, whereby administration of 18 h of intraperitoneal lipopolysaccharide (LPS; 5 mg/kg of body weight) was followed by intratrachealEscherichia coli(106CFU) in wild-type mice or those lacking hepatocyte STAT3 (hepSTAT3−/−). Pneumonia alone (without endotoxemia) was effectively controlled in the absence of liver STAT3. Following endotoxemia and pneumonia, however, hepSTAT3−/−mice, with significantly reduced levels of circulating and airspace acute-phase proteins, exhibited significantly elevated lung and blood bacterial burdens and mortality. These data suggested that STAT3-dependent liver responses are necessary to promote host defense. While neither recruited airspace neutrophils nor lung injury was altered in endotoxemic hepSTAT3−/−mice, alveolar macrophage reactive oxygen species generation was significantly decreased. Additionally, bronchoalveolar lavage fluid from this group of hepSTAT3−/−mice allowed greater bacterial growthex vivo. These results suggest that hepatic STAT3 activation promotes both cellular and humoral lung defenses. Taken together, induction of liver STAT3-dependent gene expression programs is essential to countering the deleterious consequences of sepsis on pneumonia susceptibility.
Propionibacterium acnes Recovered from Atherosclerotic Human Carotid Arteries Undergoes Biofilm Dispersion and Releases Lipolytic and Proteolytic Enzymes in Response to Norepinephrine Challenge
In Vitro
Authors: Lanter Bernard B. , Davies David G.
Published: July 2015

In the present study, human atherosclerotic carotid arteries were examined following endarterectomy for the presence of the Gram-positive bacterium
Propionibacterium acnes
and its potential association with biofilm structures within the arterial wall. The
P. acnes
16S rRNA gene was detectable in 4 of 15 carotid artery samples, and viable
P. acnes
was one among 10 different bacterial species recoverable in culture. Fluorescence
in situ
hybridization analysis of 5 additional atherosclerotic carotid arteries demonstrated biofilm bacteria within all samples, with
P. acnes
detectable in 4 samples. We also demonstrated that laboratory-grown cultures of
P. acnes
biofilms were susceptible to induction of a biofilm dispersion response when challenged with physiologically relevant levels of norepinephrine in the presence of iron-bound transferrin or with free iron. The production and release of lipolytic and proteolytic extracellular enzymes by
P. acnes
were shown to increase in iron-induced dispersed biofilms, and these dispersion-induced
P. acnes
VP1 biofilms showed increased expression of mRNAs for the triacylglycerol lipases PPA2105 and PPA1796 and the hyaluronate lyase PPA380 compared to that in untreated biofilms. These results demonstrate that
P. acnes
can infect the carotid arteries of humans with atherosclerosis as a component of multispecies biofilms and that dispersion is inducible for this organism, at least
in vitro
, with physiologically relevant levels of norepinephrine resulting in the production and release of degradative enzymes.
Acylation Enhances, but Is Not Required for, the Cytotoxic Activity of Mannheimia haemolytica Leukotoxin in Bighorn Sheep
Authors: Batra Sai A. , Shanthalingam Sudarvili , Munske Gerhard R. , Raghavan Bindu , Kugadas Abirami , Bavanthasivam Jegarubee , Highlander Sarah K. , Srikumaran Subramaniam
Published: July 2015

Mannheimia haemolytica
causes pneumonia in domestic and wild ruminants. Leukotoxin (Lkt) is the most important virulence factor of the bacterium. It is encoded within the four-gene
lktCABD
operon:
lkt
A encodes the structural protoxin, and
lktC
encodes a
trans
-acylase that adds fatty acid chains to internal lysine residues in the protoxin, which is then secreted from the cell by a type 1 secretion system apparatus encoded by
lktB
and
lktD
. It has been reported that LktC-mediated acylation is necessary for the biological effects of the toxin. However, an LktC mutant that we developed previously was only partially attenuated in its virulence for cattle. The objective of this study was to elucidate the role of LktC-mediated acylation in Lkt-induced cytotoxicity. We performed this study in bighorn sheep (
Ovis canadensis
) (BHS), since they are highly susceptible to
M. haemolytica
infection. The LktC mutant caused fatal pneumonia in 40% of inoculated BHS. On necropsy, a large number of necrotic polymorphonuclear leukocytes (PMNs) were observed in the lungs. Lkt from the mutant was cytotoxic to BHS PMNs in an
in vitro
cytotoxicity assay. Flow cytometric analysis of mutant Lkt-treated PMNs revealed the induction of necrosis. Scanning electron microscopic analysis revealed the presence of pores and blebs on mutant-Lkt-treated PMNs. Mass spectrometric analysis confirmed that the mutant secreted an unacylated Lkt. Taken together, these results suggest that acylation is not necessary for the cytotoxic activity of
M. haemolytica
Lkt but that it enhances the potency of the toxin.
The Aggregatibacter actinomycetemcomitans Cytolethal Distending Toxin Active Subunit CdtB Contains a Cholesterol Recognition Sequence Required for Toxin Binding and Subunit Internalization
Authors: Boesze-Battaglia Kathleen , Walker Lisa P. , Zekavat Ali , Dlakić Mensur , Scuron Monika Damek , Nygren Patrik , Shenker Bruce J.
Published: July 2015

Induction of cell cycle arrest in lymphocytes following exposure to theAggregatibacter actinomycetemcomitanscytolethal distending toxin (Cdt) is dependent upon the integrity of lipid membrane microdomains. Moreover, we have previously demonstrated that the association of Cdt with target cells involves the CdtC subunit which binds to cholesterol via a cholesterol recognition amino acid consensus sequence (CRAC site). In this study, we demonstrate that the active Cdt subunit, CdtB, also is capable of binding to large unilamellar vesicles (LUVs) containing cholesterol. Furthermore, CdtB binding to cholesterol involves a similar CRAC site as that demonstrated for CdtC. Mutation of the CRAC site reduces binding to model membranes as well as toxin binding and CdtB internalization in both Jurkat cells and human macrophages. A concomitant reduction in Cdt-induced toxicity was also noted, indicated by reduced cell cycle arrest and apoptosis in Jurkat cells and a reduction in the proinflammatory response in macrophages (interleukin 1β [IL-1β] and tumor necrosis factor alpha [TNF-α] release). Collectively, these observations indicate that membrane cholesterol serves as an essential ligand for both CdtC and CdtB and, further, that this binding is necessary for both internalization of CdtB and subsequent molecular events leading to intoxication of cells.