bluuebunny/crossref_metadata_2025_split huggingface dataset:
Pre‐exposure to adenosine, acting via A2A receptors on endothelial cells, alters the protein kinase A dependence of adenosine‐induced dilation in skeletal muscle resistance arterioles
Authors: Maimon Nir , Titus Patricia A. , Sarelius Ingrid H. 
Published: April 2014

Key points
The adenosine‐dependent component of functional dilation of small resistance arterioles acts via A2A receptors located on endothelium to activate KATP channels on associated vascular smooth muscle.
A2A receptors are Gs‐coupled, hence receptor occupancy should activate cAMP/protein kinase A (PKA) cell signalling pathways However, pre‐exposure to adenosine alters the PKA dependence of the response and renders the vessel insensitive to PKA inhibition.
The adenosine pre‐exposure effect is mimicked by pre‐activation of PKA and is specific to adenosine, as the PKA dependence of dilation to isoproterenol (another Gs‐coupled agonist) is not affected by pre‐exposure.
Activation of PKA alone does not induce dilation. An additional signalling mechanism, dependent on increased EC Ca2+ via activation of cyclic nucleotide gated channels, is required together with PKA activation to produce A2A‐dependent vasodilation.
This novel identification of variability in signalling downstream from a single receptor to produce the same response may reflect a mechanism for integration of key homeostatic responses.
AbstractAdenosine (ADO) is an endogenous vasodilatory purine widely recognized to be a significant contributor to functional hyperaemia. Despite this, many aspects of the mechanisms by which ADO induces dilation in small resistance arterioles are not established, or appear contradictory. These include: identification of the primary receptor subtype; its location on endothelial (EC) or vascular smooth muscle cells; whether ADO acts on KATP channels in these resistance vessels; and the contribution of cAMP/protein kinase A (PKA) signalling to the response. In intravital microscopy studies of intact or EC‐denuded skeletal muscle arterioles, we show that ADO acts via A2A receptors located on ECs to produce vasodilation via activation of KATP channels located on vascular smooth muscle cells. Importantly, we found that the signalling pathway involves cAMP as expected, but that a requirement for PKA activation is demonstrable only if the vessel is not pre‐exposed to ADO. That is, PKA‐dependent signalling varies with pre‐exposure to ADO. Further, we show that PKA activation alone is not sufficient to dilate these arterioles; an additional EC calcium‐dependent signalling mechanism is required for vasodilation to ADO. The ability of arterioles in situ to respond to occupancy of a specific receptor by utilizing different cell signalling pathways under different conditions to produce the same response allows the arteriole to respond to key homeostatic requirements using more than a single signalling mechanism. Clearly, this is likely to be physiologically advantageous, but the role for this signalling flexibility in the integrated arteriolar response that underlies functional hyperaemia will require further exploration.
Antifungal Susceptibility Patterns of Opportunistic Fungi in the Genera Verruconis and Ochroconis
Authors: Seyedmousavi S. , Samerpitak K. , Rijs A. J. M. M. , Melchers W. J. G. , Mouton J. W. , Verweij P. E. , de Hoog G. S. 
Published: April 2014

ABSTRACT

Species of
Verruconis
and species of
Ochroconis
are dematiaceous fungi generally found in the environment but having the ability to infect humans, dogs, cats, poultry, and fish. This study presents the antifungal susceptibility patterns of these fungi at the species level. Forty strains originating from clinical and environmental sources were phylogenetically identified at the species level by using sequences of the ribosomal DNA internal transcribed spacer (rDNA ITS).
In vitro
antifungal susceptibility testing was performed against eight antifungals, using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method. The geometric mean MICs for amphotericin B (AMB), flucytosine (5FC), fluconazole (FLC), itraconazole (ITC), voriconazole (VRC), and posaconazole (POS) and minimum effective concentrations (MECs) for caspofungin (CAS) and anidulafungin (AFG) across the
Ochroconis
and
Verruconis
species were as follows, in increasing order. For
Verruconis
species, the values (μg/ml) were as follows: AFG, 0.04; POS, 0.25; ITC, 0.37; AMB, 0.50; CAS, 0.65; VRC, 0.96; 5FC, 10.45; and FLC, 47.25. For
Ochroconis
species, the values (μg/ml) were as follows: AFG, 0.06; POS, 0.11; CAS, 0.67; VRC, 2.76; ITC, 3.94; AMB, 5.68; 5FC, 34.48; and FLC, 61.33. Antifungal susceptibility of
Ochroconis
and
Verruconis
was linked with phylogenetic distance and thermotolerance. Echinocandins and POS showed the greatest
in vitro
activity, providing possible treatment options for
Ochroconis
and
Verruconis
infections.
Population Pharmacokinetic/Pharmacodynamic Analysis of the Bactericidal Activities of Sutezolid (PNU-100480) and Its Major Metabolite against Intracellular Mycobacterium tuberculosis in
Ex Vivo
Whole-Blood Cultures of Patients with Pulmonary Tuberculosis
Authors: Zhu Tong , Friedrich Sven O. , Diacon Andreas , Wallis Robert S. 
Published: April 2014

ABSTRACT

Sutezolid (PNU-100480 [U-480]) is an oxazolidinone antimicrobial being developed for the treatment of tuberculosis. An active sulfoxide metabolite (PNU-101603 [U-603]), which reaches concentrations in plasma several times those of the parent, has been reported to drive the killing of extracellular
Mycobacterium tuberculosis
by sutezolid in hollow-fiber culture. However, the relative contributions of the parent and metabolite against intracellular
M. tuberculosis
in vivo
are not fully understood. The relationships between the plasma concentrations of U-480 and U-603 and intracellular whole-blood bactericidal activity (WBA) in
ex vivo
cultures were examined using a direct competitive population pharmacokinetic (PK)/pharmacodynamic 4-parameter sigmoid model. The data set included 690 PK determinations and 345 WBA determinations from 50 tuberculosis patients enrolled in a phase 2a sutezolid trial. The model parameters were solved iteratively. The median U-603/U-480 concentration ratio was 7.1 (range, 1 to 28). The apparent 50% inhibitory concentration of U-603 for intracellular
M. tuberculosis
was 17-fold greater than that of U-480 (90% confidence interval [CI], 9.9- to 53-fold). Model parameters were used to simulate
in vivo
activity after oral dosing with sutezolid at 600 mg twice a day (BID) and 1,200 mg once a day (QD). Divided dosing resulted in greater cumulative activity (−0.269 log
10
per day; 90% CI, −0.237 to −0.293 log
10
per day) than single daily dosing (−0.186 log
10
per day; 90% CI, −0.160 to −0.208 log
10
per day). U-480 accounted for 84% and 78% of the activity for BID and QD dosing, respectively, despite the higher concentrations of U-603. Killing of intracellular
M. tuberculosis
by orally administered sutezolid is mainly due to the activity of the parent compound. Taken together with the findings of other studies in the hollow-fiber model, these findings suggest that sutezolid and its metabolite act on different mycobacterial subpopulations.
Altered Pharmacokinetics of Piperacillin in Febrile Neutropenic Patients with Hematological Malignancy
Authors: Sime Fekade Bruck , Roberts Michael S. , Warner Morgyn S. , Hahn Uwe , Robertson Thomas A. , Yeend Sue , Phay Andy , Lehman Sheila , Lipman Jeffrey , Peake Sandra L. , Roberts Jason A. 
Published: April 2014

ABSTRACT
This study assessed the pharmacokinetics and dosing adequacy of piperacillin in febrile neutropenic patients after the first dose. Pharmacokinetic analysis was performed using noncompartmental methods. We observed an elevated volume of distribution (29.7 ± 8.0 liters [mean ± standard deviation]) and clearance (20.2 ± 7.5 liters/h) compared to data from other patient populations. Antibiotic exposure did not consistently result in therapeutic targets. We conclude that alternative dosing strategies guided by therapeutic drug monitoring may be required to optimize exposure.
In Vitro
and
In Vivo
Activities of Tigecycline-Colistin Combination Therapies against Carbapenem-Resistant Enterobacteriaceae
Authors: Betts Jonathan W. , Phee Lynette M. , Hornsey Michael , Woodford Neil , Wareham David W. 
Published: April 2014

ABSTRACT

We assessed the activity of tigecycline (TGC) combined with colistin (COL) against carbapenem-resistant enterobacteria. Synergy occurred
in vitro
against the majority of isolates, with the exception of
Serratia marcescens
. In a simple animal model (
Galleria mellonella
), TGC-COL was superior (
P
< 0.01) in treating
Escherichia coli
,
Klebsiella pneumoniae
, and
Enterobacter
infections, including those with TGC-COL resistance. Clinical studies are needed to determine whether TGC-COL regimens may be a viable option.
Role of Capsule and Suilysin in Mucosal Infection of Complement-Deficient Mice with Streptococcus suis
Authors: Seitz Maren , Beineke Andreas , Singpiel Alena , Willenborg Jörg , Dutow Pavel , Goethe Ralph , Valentin-Weigand Peter , Klos Andreas , Baums Christoph G. 
Published: April 2014

ABSTRACT

Virulent
Streptococcus suis
serotype 2 strains are invasive extracellular bacteria causing septicemia and meningitis in piglets and humans. One objective of this study was to elucidate the function of complement in innate immune defense against
S. suis
. Experimental infection of wild-type (WT) and C3
−/−
mice demonstrated for the first time that the complement system protects naive mice against invasive mucosal
S. suis
infection.
S. suis
WT but not an unencapsulated mutant caused mortality associated with meningitis and other pathologies in C3
−/−
mice. The capsule contributed also substantially to colonization of the upper respiratory tract. Experimental infection of C3
−/−
mice with a suilysin mutant indicated that suilysin expression facilitated an early disease onset and the pathogenesis of meningitis. Flow cytometric analysis revealed C3 antigen deposition on the surface of ca. 40% of
S. suis
WT bacteria after opsonization with naive WT mouse serum, although to a significantly lower intensity than on the unencapsulated mutant.
Ex vivo
multiplication in murine WT and C3
−/−
blood depended on capsule but not suilysin expression. Interestingly,
S. suis
invasion of inner organs was also detectable in C5aR
−/−
mice, suggesting that chemotaxis and activation of immune cells via the anaphylatoxin receptor C5aR is, in addition to opsonization, a further important function of the complement system in defense against mucosal
S. suis
infection. In conclusion, we unequivocally demonstrate here the importance of complement against mucosal
S. suis
serotype 2 infection and that the capsule of this pathogen is also involved in escape from complement-independent immunity.
Phosphoethanolamine Decoration of Neisseria gonorrhoeae Lipid A Plays a Dual Immunostimulatory and Protective Role during Experimental Genital Tract Infection
Authors: Packiam Mathanraj , Yedery Roshan D. , Begum Afrin A. , Carlson Russell W. , Ganguly Jhuma , Sempowski Gregory D. , Ventevogel Melissa S. , Shafer William M. , Jerse Ann E. 
Published: April 2014

ABSTRACT

The induction of an intense inflammatory response by
Neisseria gonorrhoeae
and the persistence of this pathogen in the presence of innate effectors is a fascinating aspect of gonorrhea. Phosphoethanolamine (PEA) decoration of lipid A increases gonococcal resistance to complement-mediated bacteriolysis and cationic antimicrobial peptides (CAMPs), and recently we reported that wild-type
N. gonorrhoeae
strain FA1090 has a survival advantage relative to a PEA transferase A (
lptA
) mutant in the human urethral-challenge and murine lower genital tract infection models. Here we tested the immunostimulatory role of this lipid A modification. Purified lipooligosaccharide (LOS) containing lipid A devoid of the PEA modification and an
lptA
mutant of strain FA19 induced significantly lower levels of NF-κB in human embryonic kidney Toll-like receptor 4 (TLR4) cells and murine embryonic fibroblasts than wild-type LOS of the parent strain. Moreover, vaginal proinflammatory cytokines and chemokines were not elevated in female mice infected with the isogenic
lptA
mutant, in contrast to mice infected with the wild-type and complemented
lptA
mutant bacteria. We also demonstrated that
lptA
mutant bacteria were more susceptible to human and murine cathelicidins due to increased binding by these peptides and that the differential induction of NF-κB by wild-type and unmodified lipid A was more pronounced in the presence of CAMPs. This work demonstrates that PEA decoration of lipid A plays both protective and immunostimulatory roles and that host-derived CAMPs may further reduce the capacity of PEA-deficient lipid A to interact with TLR4 during infection.
ALOX12
in Human Toxoplasmosis
Authors: Witola William H. , Liu Susan Ruosu , Montpetit Alexandre , Welti Ruth , Hypolite Magali , Roth Mary , Zhou Ying , Mui Ernest , Cesbron-Delauw Marie-France , Fournie Gilbert J. , Cavailles Pierre , Bisanz Cordelia , Boyer Kenneth , Withers Shawn , Noble A. Gwendolyn , Swisher Charles N. , Heydemann Peter T. , Rabiah Peter , Muench Stephen P. , McLeod Rima 
Published: April 2014

ABSTRACT

ALOX12
is a gene encoding arachidonate 12-lipoxygenase (12-LOX), a member of a nonheme lipoxygenase family of dioxygenases.
ALOX12
catalyzes the addition of oxygen to arachidonic acid, producing 12-hydroperoxyeicosatetraenoic acid (12-HPETE), which can be reduced to the eicosanoid 12-HETE (12-hydroxyeicosatetraenoic acid). 12-HETE acts in diverse cellular processes, including catecholamine synthesis, vasoconstriction, neuronal function, and inflammation. Consistent with effects on these fundamental mechanisms, allelic variants of
ALOX12
are associated with diseases including schizophrenia, atherosclerosis, and cancers, but the mechanisms have not been defined.
Toxoplasma gondii
is an apicomplexan parasite that causes morbidity and mortality and stimulates an innate and adaptive immune inflammatory reaction. Recently, it has been shown that a gene region known as
Toxo1
is critical for susceptibility or resistance to
T. gondii
infection in rats. An orthologous gene region with
ALOX12
centromeric is also present in humans. Here we report that the human
ALOX12
gene has susceptibility alleles for human congenital toxoplasmosis (rs6502997 [
P
, <0.000309], rs312462 [
P
, <0.028499], rs6502998 [
P
, <0.029794], and rs434473 [
P
, <0.038516]). A human monocytic cell line was genetically engineered using lentivirus RNA interference to knock down
ALOX12
. In
ALOX12
knockdown cells,
ALOX12
RNA expression decreased and levels of the
ALOX12
substrate, arachidonic acid, increased.
ALOX12
knockdown attenuated the progression of
T. gondii
infection and resulted in greater parasite burdens but decreased consequent late cell death of the human monocytic cell line. These findings suggest that
ALOX12
influences host responses to
T. gondii
infection in human cells.
ALOX12
has been shown in other studies to be important in numerous diseases. Here we demonstrate the critical role
ALOX12
plays in
T. gondii
infection in humans.
Inhibition of the Alternative Pathway of Nonhuman Infant Complement by Porin B2 Contributes to Virulence of Neisseria meningitidis in the Infant Rat Model
Authors: Lewis Lisa A. , Vu David M. , Granoff Dan M. , Ram Sanjay 
Published: April 2014

ABSTRACT

Neisseria meningitidis
utilizes capsular polysaccharide, lipooligosaccharide (LOS) sialic acid, factor H binding protein (fHbp), and neisserial surface protein A (NspA) to regulate the alternative pathway (AP) of complement. Using meningococcal mutants that lacked all four of the above-mentioned molecules (quadruple mutants), we recently identified a role for PorB2 in attenuating the human AP; inhibition was mediated by human fH, a key downregulatory protein of the AP. Previous studies showed that fH downregulation of the AP via fHbp or NspA is specific for human fH. Here, we report that PorB2-expressing quadruple mutants also regulate the AP of baby rabbit and infant rat complement. Blocking a human fH binding region on PorB2 of the quadruple mutant of strain 4243 with a chimeric protein that comprised human fH domains 6 and 7 fused to murine IgG Fc enhanced AP-mediated baby rabbit C3 deposition, which provided evidence for an fH-dependent mechanism of nonhuman AP regulation by PorB2. Using isogenic mutants of strain H44/76 that differed only in their PorB molecules, we confirmed a role for PorB2 in resistance to killing by infant rat serum. The PorB2-expressing strain also caused higher levels of bacteremia in infant rats than its isogenic PorB3-expressing counterpart, thus providing a molecular basis for increased survival of PorB2 isolates in this model. These studies link PorB2 expression with infection of infant rats, which could inform the choice of meningococcal strains for use in animal models, and reveals, for the first time, that PorB2-expressing strains of
N. meningitidis
regulate the AP of baby rabbits and rats.
Phase Variation Mediates Reductions in Expression of Surface Proteins during Persistent Meningococcal Carriage
Authors: Alamro Mohamed , Bidmos Fadil A. , Chan Hannah , Oldfield Neil J. , Newton Emma , Bai Xilian , Aidley Jack , Care Rory , Mattick Claire , Turner David P. J. , Neal Keith R. , Ala'Aldeen Dlawer A. A. , Feavers Ian , Borrow Ray , Bayliss Christopher D. 
Published: April 2014

ABSTRACT

Asymptomatic and persistent colonization of the upper respiratory tract by
Neisseria meningitidis
occurs despite elicitation of adaptive immune responses against surface antigens. A putative mechanism for facilitating host persistence of this bacterial commensal and pathogen is alterations in expression of surface antigens by simple sequence repeat (SSR)-mediated phase variation. We investigated how often phase variation occurs during persistent carriage by analyzing the SSRs of eight loci in multiple isolates from 21 carriers representative of 1 to 6 months carriage. Alterations in repeat number were detected by a GeneScan analysis and occurred at 0.06 mutations/gene/month of carriage. The expression states were determined by Western blotting and two genes,
fetA
and
nadA
, exhibited trends toward low expression states. A critical finding from our unique examination of combinatorial expression states, “phasotypes,” was for significant reductions in expression of multiple phase-variable surface proteins during persistent carriage of some strains. The immune responses in these carriers were examined by measuring variant-specific PorA IgG antibodies, capsular group Y IgG antibodies and serum bactericidal activity in concomitant serum samples. Persistent carriage was associated with high levels of specific IgG antibodies and serum bactericidal activity while recent strain acquisition correlated with a significant induction of antibodies. We conclude that phase-variable genes are driven into lower expression states during long-term persistent meningococcal carriage, in part due to continuous exposure to antibody-mediated selection, suggesting localized hypermutation has evolved to facilitate host persistence.
Fibronectin-Binding Protein of Borrelia hermsii Expressed in the Blood of Mice with Relapsing Fever
Authors: Lewis Eric R. G. , Marcsisin Renee A. , Campeau Miller Shelley A. , Hue Fong , Phillips April , AuCoin David P. , Barbour Alan G. 
Published: April 2014

ABSTRACT

To identify and characterize surface proteins expressed by the relapsing fever (RF) agent
Borrelia hermsii
in the blood of infected mice, we used a cell-free filtrate of their blood to immunize congenic naive mice. The resultant antiserum was used for Western blotting of cell lysates, and gel slices corresponding to reactive bands were subjected to liquid chromatography-tandem mass spectrometry, followed by a search of the proteome database with the peptides. One of the immunogens was identified as the BHA007 protein, which is encoded by a 174-kb linear plasmid. BHA007 had sequence features of lipoproteins, was surface exposed by the criteria of
in situ
protease susceptibility and agglutination of Vtp
−
cells by anti-BHA007 antibodies, and was not essential for
in vitro
growth. BHA007 elicited antibodies during experimental infection of mice, but immunization with recombinant protein did not confer protection against needle-delivered infection. Open reading frames (ORFs) orthologous to BHA007 were found on large plasmids of other RF species, including the coding sequences for the CihC proteins of
Borrelia duttonii
and
B. recurrentis
, but not in Lyme disease
Borrelia
species. Recombinant BHA007 bound both human and bovine fibronectin with

K
d

(dissociation constant) values of 22 and 33 nM, respectively, and bound to C4-binding protein with less affinity. The distant homology of BHA007 and its orthologs to BBK32 proteins of Lyme disease species, as well as to previously described BBK32-like proteins in relapsing fever species, indicates that BHA007 is a member of a large family of multifunctional proteins in
Borrelia
species that bind to fibronectin as well as other host proteins.
Identification of Early Interactions between Francisella and the Host
Authors: Roberts Lydia M. , Tuladhar Shraddha , Steele Shaun P. , Riebe Kristina J. , Chen Ching-ju , Cumming R. Ian , Seay Sarah , Frothingham Richard , Sempowski Gregory D. , Kawula Thomas H. , Frelinger Jeffrey A. 
Published: April 2014

ABSTRACT

The adaptive immune response to
Francisella tularensis
is dependent on the route of inoculation. Intradermal inoculation with the
F. tularensis
live vaccine strain (LVS) results in a robust Th1 response in the lungs, whereas intranasal inoculation produces fewer Th1 cells and instead many Th17 cells. Interestingly, bacterial loads in the lungs are similar early after inoculation by these two routes. We hypothesize that the adaptive immune response is influenced by local events in the lungs, such as the type of cells that are first infected with
Francisella
. Using fluorescence-activated cell sorting, we identified alveolar macrophages as the first cell type infected in the lungs of mice intranasally inoculated with
F. novicida
U112, LVS, or
F. tularensis
Schu S4. Following bacterial dissemination from the skin to the lung, interstitial macrophages or neutrophils are infected. Overall, we identified the early interactions between
Francisella
and the host following two different routes of inoculation.