bluuebunny/crossref_metadata_2025 huggingface dataset:
Potent and Selective Peptide Agonists of α‐Melanocyte Stimulating Hormone (αMSH) Action at Human Melanocortin Receptor 5; their Synthesis and Biological Evaluation in vitro
Authors: Bednarek Maria A. , MacNeil Tanya , Tang Rui , Fong Tung M. , Angeles Cabello M. , Maroto Marta , Teran Ana
Published: May 2007

Melanocortin receptors (MC1‐5R) and their endogenous ligands (melanocyte‐stimulating hormones and adrenocorticotropic hormone) are involved in many physiological processes in humans. Of those receptors, the actions of MC5R are the least understood despite its broad presence in the numerous peripheral tissues and brain. In this study, we describe synthesis and pharmacological properties in vitro (receptor‐binding affinity and agonist activity) of several cyclic analogs of αMSH which are potent agonists at hMC5R (EC50 below 1 nm) and of enhanced receptor subtype selectivity (more than 2000‐fold versus hMC1b,3R and about 70‐ to 200‐fold versus hMC4R). These compounds are analogs of Ac‐Nle4‐cyclo[Asp5–His6‐D‐Nal(2′)7–Pip8–Trp9–Lys10]–NH2 (Pip: pipecolic acid) in which His6 has been replaced with sterically hindered amino acids. They may be useful tools in the elucidation of the MC5R role in skin disorders and in immunomodulatory and in anti‐inflammatory actions of αMSH.
Dynamic blood pressure control and middle cerebral artery mean blood velocity variability at rest and during exercise in humans
Authors: Ogoh S. , Dalsgaard M. K. , Secher N. H. , Raven P. B.
Published: May 2007

Aims:  Cardiac failure and ischaemic heart disease patients receive standard of care cardiac β1‐adrenergic blockade medication. Such medication reduces cardiac output and cerebral blood flow. It is unknown whether the β1‐adrenergic blockade‐induced reduction of cardiac output in the presence of an exercise‐induced reduction in cardiac–arterial baroreflex gain affects cerebral blood flow variability. This study evaluated the influence of cardiac output variability on beat‐to‐beat middle cerebral artery mean blood velocity (MCA Vmean) during exercise with and without cardiac β1‐adrenergic blockade.

Methods:  Eight men (22 ± 1 years; mean ± SE) performed 15 min bouts of moderate (105 ± 11 W) and heavy (162 ± 8 W) intensity cycling before and after cardio‐selective β1‐adrenergic blockade (0.15 mg kg−1 metoprolol). The relationship between changes in cardiac output or mean arterial pressure (MAP) and MCA Vmean as well as cardiac–arterial baroreflex gain were evaluated using transfer function analysis.

Results:  Both exercise intensities decreased the low frequency (LF) transfer function gain between cardiac output and MCA Vmean (P < 0.05) with no significant influence of β1‐blockade. In contrast, the LF transfer function gain between MAP and MCA Vmean remained stable also with no significant influence of metoprolol (P > 0.05). The LF transfer function gain between MAP and HR, an index of cardiac–arterial baroreflex gain, decreased from rest to heavy exercise with and without β1‐blockade (P < 0.05).

Conclusion:  These findings suggest that the exercise intensity related reduction in cardiac–arterial baroreflex function at its operating point does not influence the dynamic control of MCA Vmean, even when the ability of exercise‐induced increase in cardiac output is reduced by cardiac β1‐adrenergic blockade.
Human cardiovascular dose–response to supplemental oxygen
Authors: Bak Z. , Sjöberg F. , Rousseau A. , Steinvall I. , Janerot‐Sjoberg B.
Published: May 2007

Aim:  The aim of the study was to examine the central and peripheral cardiovascular adaptation and its coupling during increasing levels of hyperoxaemia. We hypothesized a dose‐related effect of hyperoxaemia on left ventricular performance and the vascular properties of the arterial tree.

Methods:  Oscillometrically calibrated arterial subclavian pulse trace data were combined with echocardiographic recordings to obtain non‐invasive estimates of left ventricular volumes, aortic root pressure and flow data. For complementary vascular parameters and control purposes whole‐body impedance cardiography was applied. In nine (seven males) supine, resting healthy volunteers, aged 23–48 years, data was collected after 15 min of air breathing and at increasing transcutaneous oxygen tensions (20, 40 and 60 kPa), accomplished by a two group, random order and blinded hyperoxemic protocol.

Results:  Left ventricular stroke volume [86 ± 13 to 75 ± 9 mL (mean ± SD)] and end‐diastolic area (19.3 ± 4.4 to 16.8 ± 4.3 cm2) declined (P < 0.05), and showed a linear, negative dose–response relationship to increasing arterial oxygen levels in a regression model. Peripheral resistance and characteristic impedance increased in a similar manner. Heart rate, left ventricular fractional area change, end‐systolic area, mean arterial pressure, arterial compliance or carbon dioxide levels did not change.

Conclusion:  There is a linear dose–response relationship between arterial oxygen and cardiovascular parameters when the systemic oxygen tension increases above normal. A direct effect of supplemental oxygen on the vessels may therefore not be excluded. Proximal aortic and peripheral resistance increases from hyperoxaemia, but a decrease of venous return implies extra cardiac blood‐pooling and compensatory relaxation of the capacitance vessels.
New Constraints on Ages of Glasses Proposed as Reference Materials for Fission‐Track Dating
Authors: Laurenzi Marinella A. , Balestrieri Maria Laura , Bigazzi Giulio , Hadler Neto Julio C. , Iunes Pedro J. , Norelli Pio , Oddone Massimo , Osorio Araya Ana Maria , Viramonte José G.
Published: May 2007

New analyses have been performed in order to enhance the data‐set on the independent ages of four glasses that have been proposed as reference materials for fission‐track dating. The results are as follows. Moldavite ‐ repeated 40Ar/39Ar age determinations on samples from deposits from Bohemia and Moravia yielded an average of 14.34 ± 0.08 Ma. This datum agrees with other recent determinations and is significantly younger than the 40Ar/39Ar age of 15.21 ± 0.15 Ma determined in the early 1980s. Macusanite (Peru) ‐four K‐Ar ages ranging from 5.44 ± 0.06 to 5.72 ± 0.12 Ma have been published previously. New 40Ar/39Ar ages gave an average of 5.12 ± 0.04 Ma. Plateau fission‐track ages determined using the IRMM‐540 certified glass and U and Th thin films for neutron fluence measurements agree better with these new 40Ar/39Ar ages than the previously published ages. Roccastrada glass (Italy) ‐ a new 40Ar/39Ar age, 2.45 ± 0.04 Ma, is consistent with previous determinations. The Quiron obsidian (Argentina) is a recently discovered glass that has been proposed as an additional reference material for its high spontaneous track density (around 100 000 cm‐2). Defects that might produce “spurious” tracks are virtually absent. An independent 40Ar/39Ar age of 8.77 ± 0.09 Ma was determined and is recommended for this glass. We believe that these materials, which will be distributed upon request to fission‐track groups, will be very useful for testing system calibrations and experimental procedures.
Recombinant
Mycobacterium bovis
Bacillus Calmette-Guérin Elicits Human Immunodeficiency Virus Type 1 Envelope-Specific T Lymphocytes at Mucosal Sites
Authors: Yu Jae-Sung , Peacock James W. , Jacobs William R. , Frothingham Richard , Letvin Norman L. , Liao Hua-Xin , Haynes Barton F.
Published: May 2007

A successful vaccine vector for human immunodeficiency virus type 1 (HIV-1) should induce anti-HIV-1 T-cell immune responses at mucosal sites. We have constructed recombinant
Mycobacterium bovis
bacillus Calmette-Guérin (rBCG) expressing an HIV-1 group M consensus envelope (Env) either as a surface, intracellular, or secreted protein as an immunogen. rBCG containing HIV-1
env
plasmids engineered for secretion induced optimal Env-specific T-cell gamma interferon enzyme-linked immunospot responses in murine spleen, female reproductive tract, and lungs. While rBCG-induced T-cell responses to HIV-1 envelope in spleen were lower than those induced by adenovirus prime/recombinant vaccinia virus (rAd-rVV) boost, rBCG induced comparable responses to rAd-rVV immunization in the female reproductive tract and lungs. T-cell responses induced by rBCG were primarily CD4
+
, although rBCG alone did not induce anti-HIV-1 antibody. However, rBCG could prime for a protein boost by HIV-1 envelope protein. Thus, rBCG can serve as a vector for induction of anti-HIV-1 consensus Env cellular responses at mucosal sites.
Real-Time PCR for Determining Capsular Serotypes of
Haemophilus influenzae
Authors: Maaroufi Younes , De Bruyne Jean-Marc , Heymans Corine , Crokaert Françoise
Published: May 2007

A two-step real-time PCR assay targeting all six capsulation loci of
Haemophilus influenzae
(i.e., serotypes a to f) was developed and compared with a previously published qualitative PCR assay by using 131
H. influenzae
clinical isolates. There was a 98.5% concordance between the two tests. The sensitivity of detection of capsular type-specific reference strains of
H. influenzae
a to c (10
1
CFU/PCR) was higher than that for type e (10
3
CFU/PCR) and types d and f (10
4
CFU/PCR), and a broader dynamic range was obtained (5 to 8 log
10
units). No cross-reaction was observed with bacteria commonly isolated from the respiratory tract. We showed that both PCR assays are more reliable than slide agglutination serotyping. The real-time PCR-based assay seems to be an alternative of choice for the epidemiological follow-up of
H. influenzae
invasive infections.
Enterohepatic
Helicobacter
Species Are Prevalent in Mice from Commercial and Academic Institutions in Asia, Europe, and North America
Authors: Taylor Nancy S. , Xu Shilu , Nambiar Prashant , Dewhirst Floyd E. , Fox James G.
Published: May 2007

The discovery of
Helicobacter hepaticus
and its role in hepatitis, hepatocellular carcinoma, typhlocolitis, and lower-bowel carcinoma in murine colonies was followed by the isolation and characterization of other
Helicobacter
spp. involved in enterohepatic disease. Colonization of mouse colonies with members of the family
Helicobacteriaceae
has become an increasing concern for the research community. From 2001 to 2005, shipments of selected gift mice from other institutions and mice received from specified commercial vendors were screened for
Helicobacter
spp. by culture of cecal tissue. The identities of the isolates were confirmed by genus-specific PCR, followed by species-specific PCR and restriction fragment length polymorphism analysis. Sequencing of the 16S rRNA gene was performed if the species identity was not apparent. The survey included 79 mice from 34 sources: 2 commercial sources and 16 research sources from the United States and 1 commercial source and 15 research sources from Canada, Europe, or Asia.
Helicobacter
spp. were cultured from the ceca of 62 of 79 mice. No
Helicobacter
spp. were found in mice from advertised
Helicobacter
-free production areas from two U.S. vendors. Multiple
Helicobacter
spp. were found in mice from one vendor's acknowledged
Helicobacter
-infected production area. The European commercial vendor had mice infected with novel
Helicobacter
sp. strain MIT 96-1001. Of the U.S. academic institutions, 6 of 16 (37%) had mice infected with
Helicobacter hepaticus
; but monoinfection with
H. bilis
,
H. mastomyrinus
,
H. rodentium
, and MIT 96-1001 was also encountered, as were mice infected simultaneously with two
Helicobacter
spp. Non-U.S. academic institutions had mice that were either monoinfected with
H. hepaticus
, monoinfected with seven other
Helicobacter
spp., or infected with a combination of
Helicobacter
spp. This survey indicates that 30 of 34 (88%) commercial and academic institutions in Canada, Europe, Asia, Australia, and the United States have mouse colonies infected with
Helicobacter
spp. Mice from 20 of the 34 institutions (59%) were most commonly colonized with
H. hepaticus
alone or in combination with other
Helicobacter
spp. These results indicate that a broad range of
Helicobacter
spp. infect mouse research colonies. The potential impact of these organisms on in vivo experiments continues to be an important issue for mice being used for biomedical research.
Meningitis Due toGemella haemolysansin a Pediatric Case
Authors: Anil Murat , Ozkalay Nisel , Helvaci Mehmet , Agus Neval , Guler Ozlem , Dikerler Aysu , Kanar Berat
Published: May 2007

Gemella haemolysansis a rare pathogen in cases of bacterial meningitis. We present a case of meningitis due toG. haemolysansin a 17-month-old boy. This is the first reported case ofGemellameningitis in a child. The patient completely recovered following intravenous therapy with linezolid and chloramphenicol.
Simultaneous Detection and High-Throughput Identification of a Panel of RNA Viruses Causing Respiratory Tract Infections
Authors: Li Haijing , McCormac Melinda A. , Estes R. Wray , Sefers Susan E. , Dare Ryan K. , Chappell James D. , Erdman Dean D. , Wright Peter F. , Tang Yi-Wei
Published: May 2007

Clinical presentations for viral respiratory tract infections are often nonspecific, and a rapid, high-throughput laboratory technique that can detect a panel of common viral pathogens is clinically desirable. We evaluated two multiplex reverse transcription-PCR (RT-PCR) products coupled with microarray-based systems for simultaneous detection of common respiratory tract viral pathogens. The NGEN respiratory virus analyte-specific assay (Nanogen, San Diego, CA) detects influenza A virus (Flu-A) and Flu-B, parainfluenza virus 1 (PIV-1), PIV-2, and PIV-3, and respiratory syncytial virus (RSV), while the ResPlex II assay (Genaco Biomedical Products, Inc., Huntsville, AL) detects Flu-A, Flu-B, PIV-1, PIV-2, PIV-3, PIV-4, RSV, human metapneumovirus (hMPV), rhinoviruses (RhVs), enteroviruses (EnVs), and severe acute respiratory syndrome (SARS) coronavirus (CoV). A total of 360 frozen respiratory specimens collected for a full year were tested, and results were compared to those obtained with a combined reference standard of cell culture and monoplex real-time TaqMan RT-PCR assays. NGEN and ResPlex II gave comparable sensitivities for Flu-A (82.8 to 86.2%), Flu-B (90.0 to 100.0%), PIV-1 (87.5 to 93.8%), PIV-3 (66.7 to 72.2%), and RSV (63.3 to 73.3%); both assays achieved excellent specificities (99.1 to 100.0%) for these five common viruses. The ResPlex II assay detected hMPV in 13 (3.6%) specimens, with a sensitivity of 80.0% and specificity of 99.7%. The ResPlex II assay also differentiated RSV-A and RSV-B and gave positive results for RhV and EnV in 31 (8.6%) and 19 (5.3%) specimens, respectively. PIV-2, PIV-4, and SARS CoV were not detected in the specimens tested. The two systems can process 80 (NGEN) and 96 (ResPlex II) tests per run, with a hands-on time of approximately 60 min and test turnaround times of 6 h (ResPlex II) and 9 h (NGEN). Multiple-panel testing detected an additional unsuspected 9 (3.4%) PIV-1 and 10 (3.7%) PIV-3 infections. While test sensitivities for RSV and PIV-3 need improvement, both the NGEN and ResPlex II assays provide user-friendly and high-throughput tools for simultaneous detection and identification of a panel of common respiratory viral pathogens in a single test format. The multiplex approach enhances diagnosis through detection of respiratory viral etiologic agents in cases in which the presence of the agent was not suspected and a test was not ordered by the clinicians.
Molecular Epidemiology of Rotavirus in Central and Southeastern Europe
Authors: Tcheremenskaia Olga , Marucci Gianluca , De Petris Simona , Ruggeri Franco Maria , Dovecar Darja , Sternak Suncanica Ljubin , Matyasova Irena , Dhimolea Majlinda Kota , Mladenova Zornitsa , Fiore Lucia
Published: May 2007

A surveillance network was implemented by the Istituto Superiore di Sanità of Rome in collaboration with laboratories of virology in Czech Republic, Slovenia, Croatia, Albania, and Bulgaria. About 1,500 rotavirus-positive stool samples were collected from children with severe gastroenteritis admitted to hospitals or outpatient wards between 2004 and 2006. The G and P genotypes were determined by reverse transcription-nested PCR. Significant differences were found in the geographical distributions of rotavirus genotypes between countries participating in the study. The prevalence of “common” G/P combinations, G1P[8], G3P[8], G4P[8], and G2P[4], ranged between 50 and 85%. The G9 genotype, which is emerging worldwide, was identified in 2 to 35% of all samples depending on the country. Unusual combinations, such as G1 or G4 associated with P[4] or G2 with P[8], which may have arisen by reassortment between human strains, were found in samples from 3 to 20% of patients. The uncommon genotypes G8P[8] and G10P[6], which may have an animal origin, were also identified. Double infections with two rotavirus strains were observed in between 1.7 and 14% of cases studied. Our findings might implicate challenges for rotavirus vaccine implementation in a wide geographic area of the Balkans and Central-Eastern Europe and underscore the importance of extensive strain surveillance for success in vaccine development.
Contribution of Polymorphisms in
ankA
,
gltA
, and
groESL
in Defining Genetic Variants of
Anaplasma phagocytophilum
Authors: Shukla Sanjay K. , Aswani Vijay , Stockwell Patrick J. , Reed Kurt D.
Published: May 2007

Analysis of several nucleotide polymorphisms in polymorphic genes (
ankA
,
gltA
, and
groESL
) from 16S rRNA gene-based genetic variants of
Anaplasma phagocytophilum
from dogs in the western United States defined at least two sets of multigene polymorphisms to further characterize these variants. The multigene polymorphism approach holds promise for development of a genotyping scheme for this important pathogen.
First Insight into the Population Structure of
Mycobacterium tuberculosis
in Saudi Arabia
Authors: Al-Hajoj Sahal A. M. , Zozio Thierry , Al-Rabiah Fahad , Mohammad Viquar , Al-Nasser Maryam , Sola Christophe , Rastogi Nalin
Published: May 2007

This study constitutes a first attempt to describe the genetic population structure and drug resistance of the tubercle bacilli circulating in Saudi Arabia. A total of 1,505 clinical isolates of
M. tuberculosis
, isolated between 2002 and 2005 from seven regions of Saudi Arabia, were studied. The sample studied showed a male-to-female sex ratio of 1.27, with half of the cases among foreign-born individuals and 47% within the 21- to 40-year-old age group; a total resistance rate of 19.7%; and multiple drug resistance of 4.5%. Upon spoligotyping, a total of 387 individual patterns were obtained (clustering rate, 86.4%; 182 clusters containing between 2 and 130 isolates per cluster). A total of 94% of the strains matched the spoligotype patterns in an international database. Nearly 81% of the isolates in this study belonged to established phylogeographic clades: Central Asian (CAS), 22.5%; ill-defined T clade, 19.5%; East African-Indian (EAI), 13.5%; Haarlem, 7.5%; Latin American-Mediterranean, 7.2%; Beijing, 4.4%; Manu, 2.7%; X, 0.9%; and Bovis, 0.9%. Two clonal complexes with unique spoligotyping signatures (octal codes 703777707770371 and 467777377413771) specific to Saudi Arabia were identified. These belonged to the CAS and EAI clades, respectively, as confirmed upon secondary typing using mycobacterial interspersed repetitive units (MIRUs). The results obtained underline the predominance of historic clones of principal genetic group 1, which are responsible for roughly 45% of all tuberculosis cases in Saudi Arabia. The high rate of clustering observed might be an indication of rapid ongoing transmission within certain communities and/or subpopulations in Saudi Arabia; nonetheless, spoligotyping is known to overestimate clustering, and only a systematic second-line typing, such as MIRUs, coupled with a better tuberculosis registry and epidemiological investigations would allow us to know the exact rate of ongoing transmission and associated risk factors in Saudi Arabia.
Evaluation of the Colorimetric VITEK 2 Card for Identification of Gram-Negative Nonfermentative Rods: Comparison to 16S rRNA Gene Sequencing
Authors: Zbinden A. , Böttger E. C. , Bosshard P. P. , Zbinden R.
Published: May 2007

Ninety strains of a collection of well-identified clinical isolates of gram-negative nonfermentative rods collected over a period of 5 years were evaluated using the new colorimetric VITEK 2 card. The VITEK 2 colorimetric system identified 53 (59%) of the isolates to the species level and 9 (10%) to the genus level; 28 (31%) isolates were misidentified. An algorithm combining the colorimetric VITEK 2 card and 16S rRNA gene sequencing for adequate identification of gram-negative nonfermentative rods was developed. According to this algorithm, any identification by the colorimetric VITEK 2 card other than
Achromobacter xylosoxidans
,
Acinetobacter
sp.,
Burkholderia cepacia
complex,
Pseudomonas aeruginosa
, and
Stenotrophomonas maltophilia
should be subjected to 16S rRNA gene sequencing when accurate identification of nonfermentative rods is of concern.
GxxxG Motif of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Transmembrane Domain Is Not Involved in Trimerization and Is Not Important for Entry
Authors: Corver Jeroen , Broer Rene , van Kasteren Puck , Spaan Willy
Published: May 2007

Recently, a paper was published in which it was proposed that the GxxxG motif of the severe acute respiratory syndrome (SARS) coronavirus spike (S) protein transmembrane domain plays a vital role in oligomerization of the protein (E. Arbely, Z. Granot, I. Kass, J. Orly, and I. T. Arkin, Biochemistry 45:11349-11356, 2006). Here, we show that the GxxxG motif is not involved in SARS S oligomerization by trimerization analysis of S GxxxG mutant proteins. In addition, the capability of S to mediate entry of SARS S-pseudotyped particles overall was affected moderately in the mutant proteins, also arguing for a nonvital role for the GxxxG motif in SARS coronavirus entry.
High-Avidity Monoclonal Antibodies against the Human Scavenger Class B Type I Receptor Efficiently Block Hepatitis C Virus Infection in the Presence of High-Density Lipoprotein
Authors: Catanese Maria Teresa , Graziani Rita , von Hahn Thomas , Moreau Martine , Huby Thierry , Paonessa Giacomo , Santini Claudia , Luzzago Alessandra , Rice Charles M. , Cortese Riccardo , Vitelli Alessandra , Nicosia Alfredo
Published: May 2007

The human scavenger class B type 1 receptor (SR-B1/Cla1) was identified as a putative receptor for hepatitis C virus (HCV) because it binds to soluble recombinant HCV envelope glycoprotein E2 (sE2). High-density lipoprotein (HDL), a natural SR-B1 ligand, was shown to increase the in vitro infectivity of retroviral pseudoparticles bearing HCV envelope glycoproteins and of cell culture-derived HCV (HCVcc), suggesting that SR-B1 promotes viral entry in an HDL-dependent manner. To determine whether SR-B1 participates directly in HCV infection or facilitates HCV entry through lipoprotein uptake, we generated a panel of monoclonal antibodies (MAbs) against native human SR-B1. Two of them, 3D5 and C167, bound to conformation-dependent SR-B1 determinants and inhibited the interaction of sE2 with SR-B1. These antibodies efficiently blocked HCVcc infection of Huh-7.5 hepatoma cells in a dose-dependent manner. To examine the role of HDL in SR-B1-mediated HCVcc infection, we set up conditions for HCVcc production and infection in serum-free medium. HCVcc efficiently infected Huh-7.5 cells in the absence of serum lipoproteins, and addition of HDL led to a twofold increase in infectivity. However, the HDL-induced enhancement of infection had no impact on the neutralization potency of MAb C167, despite its ability to inhibit both HDL binding to cells and SR-B1-mediated lipid transfer. Of note, MAb C167 also potently blocked Huh-7.5 infection by an HCV strain recovered from HCVcc-infected chimpanzees. These results demonstrate that SR-B1 is essential for infection with HCV produced in vitro and in vivo and suggest the possible use of anti-SR-B1 antibodies as therapeutic agents.
Herpes Simplex Virus Type 1 Cytoplasmic Envelopment Requires Functional Vps4
Authors: Crump Colin M. , Yates Catherine , Minson Tony
Published: May 2007

The assembly and egress of herpesviruses are complex processes that require the budding of viral nucleocapsids into the lumen of cytoplasmic compartments to form mature infectious virus. This envelopment stage shares many characteristics with the formation of luminal vesicles in multivesicular endosomes. Through expression of dominant-negative Vps4, an enzyme that is essential for the formation of luminal vesicles in multivesicular endosomes, we now show that Vps4 function is required for the cytoplasmic envelopment of herpes simplex virus type 1. This is the first example of a large enveloped DNA virus engaging the multivesicular endosome sorting machinery to enable infectious virus production.
Insulin-Like Growth Factor II Receptor-Mediated Intracellular Retention of Cathepsin B Is Essential for Transformation of Endothelial Cells by Kaposi's Sarcoma-Associated Herpesvirus
Authors: Rose Patrick P. , Bogyo Matthew , Moses Ashlee V. , Früh Klaus
Published: May 2007

Kaposi's sarcoma-associated herpesvirus (KSHV) is the pathological agent of Kaposi's sarcoma (KS), a tumor characterized by aberrant proliferation of endothelial-cell-derived spindle cells. Since in many cancers tumorigenesis is associated with an increase in the activity of the cathepsin family, we studied the role of cathepsins in KS using an in vitro model of KSHV-mediated endothelial cell transformation. Small-molecule inhibitors and small interfering RNA (siRNA) targeting CTSB, but not other cathepsins, inhibited KSHV-induced postconfluent proliferation and the formation of spindle cells and foci of dermal microvascular endothelial cells. Interestingly, neither CTSB mRNA nor CTSB protein levels were induced in endothelial cells latently infected with KSHV. Secretion of CTSB was strongly diminished upon KSHV infection. Increased targeting of CTSB to endosomes was caused by the induction by KSHV of the expression of insulin-like growth factor-II receptor (IGF-IIR), a mannose-6-phosphate receptor (M6PR) that binds to cathepsins. Inhibition of IGF-IIR/M6PR expression by siRNA released CTSB for secretion. In contrast to the increased cathepsin secretion observed in most other tumors, viral inhibition of CTSB secretion via induction of an M6PR is crucial for the transformation of endothelial cells.
A Naturally Occurring Splice Variant of CXCL12/Stromal Cell-Derived Factor 1 Is a Potent Human Immunodeficiency Virus Type 1 Inhibitor with Weak Chemotaxis and Cell Survival Activities
Authors: Altenburg Jeffrey D. , Broxmeyer Hal E. , Jin Qingwen , Cooper Scott , Basu Sunanda , Alkhatib Ghalib
Published: May 2007

CXCL12/stromal cell-derived factor 1 is a member of the CXC family of chemokines that plays an important role in hematopoiesis and signals through CXCR4 and CXCR7. Two splice variants of human CXCL12 (CXCL12α and CXCL12β) induce chemotaxis of CXCR4
+
cells and inhibit X4 infection. Recent studies described four other novel splice variants of human CXCL12; however, their antiviral activities were not investigated. We constructed and expressed all of the CXCL12 splice variants in
Escherichia coli
. Recombinant proteins were purified through a His affinity column, and their biological properties were analyzed. All six CXCL12 variants induced chemotaxis of CXCR4
+
and CXCR7
+
cell lines. Enhancement of survival and replating capacity of human hematopoietic progenitor cells were observed with CXCL12α, CXCL12β, and CXCL12ε but not with the other variants. CXCL12γ showed the greatest antiviral activity in X4 inhibition assays and the weakest chemotaxis activity through CXCR4. The order of potency in X4 inhibition assays was as follows: CXCL12γ > CXCL12β > CXCL12α > CXCL12θ > CXCL12ε > CXCL12δ. The order of anti-human immunodeficiency virus (HIV) activity was associated with the number of BBXB motifs present in each variant; the most potent inhibitor was CXCL12γ, with five BBXB domains. The results suggest that the different C termini of CXCL12 variants may contain important molecular determinants for the observed differences in antiviral effects and other biological functions. These studies implicate CXCL12γ as a potent HIV-1 entry inhibitor with significantly reduced chemotaxis activity and small or absent effects on progenitor cell survival or replating capacity, providing important insight into the structure-function relationships of CXCL12.
Virus Subtype-Specific Features of Natural Simian Immunodeficiency Virus SIV
smm
Infection in Sooty Mangabeys
Authors: Apetrei Cristian , Gautam Rajeev , Sumpter Beth , Carter Anders C. , Gaufin Thaidra , Staprans Silvija I. , Else James , Barnes Mary , Cao Robert , Garg Seema , Milush Jeffrey M. , Sodora Donald L. , Pandrea Ivona , Silvestri Guido
Published: May 2007

Simian immunodeficiency virus (SIV) SIV
smm
naturally infects sooty mangabeys (SMs) and is the source virus of pathogenic infections with human immunodeficiency virus type 2 (HIV-2) and SIV
mac
of humans and macaques, respectively. In previous studies we characterized SIV
smm
diversity in naturally SIV-infected SMs and identified nine different phylogenetic subtypes whose genetic distances are similar to those reported for the different HIV-1 group M subtypes. Here we report that, within the colony of SMs housed at the Yerkes National Primate Research Center, at least four SIV
smm
subtypes cocirculate, with the vast majority of animals infected with SIV
smm
subtype 1, 2, or 3, resulting in the emergence of occasional recombinant forms. While SIV
smm
-infected SMs show a typically nonpathogenic course of infection, we have observed that different SIV
smm
subtypes are in fact associated with specific immunologic features. Notably, while subtypes 1, 2, and 3 are associated with a very benign course of infection and preservation of normal CD4
+
T-cell counts, three out of four SMs infected with subtype 5 show a significant depletion of CD4
+
T cells. The fact that virus replication in SMs infected with subtype 5 is similar to that in SMs infected with other SIV
smm
subtypes suggests that the subtype 5-associated CD4
+
T-cell depletion is unlikely to simply reflect higher levels of virus-mediated direct killing of CD4
+
T-cells. Taken together, this systematic analysis of the subtype-specific features of SIV
smm
infection in natural SM hosts identifies subtype-specific differences in the pathogenicity of SIV
smm
infection.
Productive Human Immunodeficiency Virus Type 1 Assembly Takes Place at the Plasma Membrane
Authors: Finzi Andrés , Orthwein Alexandre , Mercier Johanne , Cohen Éric A.
Published: May 2007

Gag proteins are necessary and sufficient to direct human immunodeficiency virus type 1 (HIV-1) particle assembly and budding. Recent evidence suggests that Gag targeting to late endosomal/multivesicular body (LE/MVB) compartments occurs prior to viral particle budding at the plasma membrane (PM). However, the route that Gag follows before reaching its steady-state destinations still remains a subject of debate. Using a subcellular fractionation method that separates PM from LE/MVB combined with pulse-chase labeling, we analyzed Gag trafficking in HIV-1-producing HEK 293T cells. Our results reveal that the majority of newly synthesized Gag is primarily targeted to the PM. While PM-targeted Gag was efficiently released, a significant fraction of the remaining cell surface-associated Gag was found to be subsequently internalized to LE/MVB, where it accumulated, thus accounting for the majority of LE/MVB-associated Gag. Importantly, this accumulation of Gag in LE/MVB was found to be cholesterol dependent since it was sensitive to the sterol-binding drugs filipin and methyl-β-cyclodextrin. These results point towards the PM as being the primary site of productive HIV-1 assembly in cells that also support Gag accumulation in intracellular compartments.
Characterization of Low-Pathogenic H5 Subtype Influenza Viruses from Eurasia: Implications for the Origin of Highly Pathogenic H5N1 Viruses
Authors: Duan L. , Campitelli L. , Fan X. H. , Leung Y. H. C. , Vijaykrishna D. , Zhang J. X. , Donatelli I. , Delogu M. , Li K. S. , Foni E. , Chiapponi C. , Wu W. L. , Kai H. , Webster R. G. , Shortridge K. F. , Peiris J. S. M. , Smith Gavin J. D. , Chen H. , Guan Y.
Published: May 2007

Highly pathogenic avian influenza (HPAI) H5N1 viruses are now endemic in many Asian countries, resulting in repeated outbreaks in poultry and increased cases of human infection. The immediate precursor of these HPAI viruses is believed to be A/goose/Guangdong/1/96 (Gs/GD)-like H5N1 HPAI viruses first detected in Guangdong, China, in 1996. From 2000 onwards, many novel reassortant H5N1 influenza viruses or genotypes have emerged in southern China. However, precursors of the Gs/GD-like viruses and their subsequent reassortants have not been fully determined. Here we characterize low-pathogenic avian influenza (LPAI) H5 subtype viruses isolated from poultry and migratory birds in southern China and Europe from the 1970s to the 2000s. Phylogenetic analyses revealed that Gs/GD-like virus was likely derived from an LPAI H5 virus in migratory birds. However, its variants arose from multiple reassortments between Gs/GD-like virus and viruses from migratory birds or with those Eurasian viruses isolated in the 1970s. It is of note that unlike HPAI H5N1 viruses, those recent LPAI H5 viruses have not become established in aquatic or terrestrial poultry. Phylogenetic analyses revealed the dynamic nature of the influenza virus gene pool in Eurasia with repeated transmissions between the eastern and western extremities of the continent. The data also show reassortment between influenza viruses from domestic and migratory birds in this region that has contributed to the expanded diversity of the influenza virus gene pool among poultry in Eurasia.
MyD88-Dependent Immune Activation Mediated by Human Immunodeficiency Virus Type 1-Encoded Toll-Like Receptor Ligands
Authors: Meier Angela , Alter Galit , Frahm Nicole , Sidhu Harlyn , Li Bin , Bagchi Aranya , Teigen Nickolas , Streeck Hendrik , Stellbrink Hans-Juergen , Hellman Judith , van Lunzen Jan , Altfeld Marcus
Published: May 2007

Immune activation is a major characteristic of human immunodeficiency virus type 1 (HIV-1) infection and a strong prognostic factor for HIV-1 disease progression. The underlying mechanisms leading to immune activation in viremic HIV-1 infection, however, are not fully understood. Here we show that, following the initiation of highly active antiretroviral therapy, the immediate decline of immune activation is closely associated with the reduction of HIV-1 viremia, which suggests a direct contribution of HIV-1 itself to immune activation. To propose a mechanism, we demonstrate that the single-stranded RNA of HIV-1 encodes multiple uridine-rich Toll-like receptor 7/8 (TLR7/8) ligands that induce strong MyD88-dependent plasmacytoid dendritic cell and monocyte activation, as well as accessory cell-dependent T-cell activation. HIV-1-encoded TLR ligands may, therefore, directly contribute to the immune activation observed during viremic HIV-1 infection. These data provide an initial rationale for inhibiting the TLR pathway to directly reduce the chronic immune activation induced by HIV-1 and the associated immune pathogenesis.
Compensatory Mutation Partially Restores Fitness and Delays Reversion of Escape Mutation within the Immunodominant HLA-B*5703-Restricted Gag Epitope in Chronic Human Immunodeficiency Virus Type 1 Infection
Authors: Crawford Hayley , Prado Julia G. , Leslie Alasdair , Hué Stéphane , Honeyborne Isobella , Reddy Sharon , van der Stok Mary , Mncube Zenele , Brander Christian , Rousseau Christine , Mullins James I. , Kaslow Richard , Goepfert Paul , Allen Susan , Hunter Eric , Mulenga Joseph , Kiepiela Photini , Walker Bruce D. , Goulder Philip J. R.
Published: May 2007

HLA-B*5703 is associated with effective immune control in human immunodeficiency virus type 1 (HIV-1) infection. Here we describe an escape mutation within the immunodominant HLA-B*5703-restricted epitope in chronic HIV-1 infection, KAFSPEVIPMF (Gag 162-172), and demonstrate that this mutation reduces viral replicative capacity. Reversion of this mutation following transmission to HLA-B*5703-negative recipients was delayed by the compensatory mutation S165N within the same epitope. These data may help explain the observed association between HLA-B*5703 and long-term control of viremia.
Surface-Exposed Adeno-Associated Virus Vp1-NLS Capsid Fusion Protein Rescues Infectivity of Noninfectious Wild-Type Vp2/Vp3 and Vp3-Only Capsids but Not That of Fivefold Pore Mutant Virions
Authors: Grieger Joshua C. , Johnson Jarrod S. , Gurda-Whitaker Brittney , Agbandje-McKenna Mavis , Samulski R. Jude
Published: May 2007

Over the past 2 decades, significant effort has been dedicated to the development of adeno-associated virus (AAV) as a vector for human gene therapy. However, understanding of the virus with respect to the functional domains of the capsid remains incomplete. In this study, the goal was to further examine the role of the unique Vp1 N terminus, the N terminus plus the recently identified nuclear localization signal (NLS) (J. C. Grieger, S. Snowdy, and R. J. Samulski, J. Virol
80
:5199-5210, 2006), and the virion pore at the fivefold axis in infection. We generated two Vp1 fusion proteins (Vp1 and Vp1NLS) linked to the 8-kDa chemokine domain of rat fractalkine (FKN) for the purpose of surface exposure upon assembly of the virion, as previously described (K. H. Warrington, Jr., O. S. Gorbatyuk, J. K. Harrison, S. R. Opie, S. Zolotukhin, and N. Muzyczka, J. Virol
78
:6595-6609, 2004). The unique Vp1 N termini were found to be exposed on the surfaces of these capsids and maintained their phospholipase A2 (PLA2) activity, as determined by native dot blot Western and PLA2 assays, respectively. Incorporation of the fusions into AAV type 2 capsids lacking a wild-type Vp1, i.e., Vp2/Vp3 and Vp3 capsid only, increased infectivity by 3- to 5-fold (Vp1FKN) and 10- to 100-fold (Vp1NLSFKN), respectively. However, the surface-exposed fusions did not restore infectivity to AAV virions containing mutations at a conserved leucine (Leu336Ala, Leu336Cys, or Leu336Trp) located at the base of the fivefold pore. EM analyses suggest that Leu336 may play a role in global structural changes to the virion directly impacting downstream conformational changes essential for infectivity and not only have local effects within the pore, as previously suggested.
DNA Immunization Using Highly Conserved Murine Cytomegalovirus Genes Encoding Homologs of Human Cytomegalovirus UL54 (DNA Polymerase) and UL105 (Helicase) Elicits Strong CD8 T-Cell Responses and Is Protective against Systemic Challenge
Authors: Morello Christopher S. , Kelley Laura A. , Munks Michael W. , Hill Ann B. , Spector Deborah H.
Published: May 2007

Human cytomegalovirus (HCMV) establishes a lifelong infection with the potential for reinfection or viral transmission even in the presence of strong and diverse CD8 T-lymphocyte responses. This suggests that the CMVs skew the host T-cell response in order to favor viral persistence. In this study, we hypothesized that the essential, nonstructural proteins that are highly conserved among the CMVs may represent a novel class of T-cell targets for vaccine-mediated protection due to their requirements for expression and sequence stability, but that the observed subdominance of these antigens in the CMV-infected host results from the virus limiting the T-cell responses to otherwise-protective specificities. We found that DNA immunization of mice with the murine CMV (MCMV) homologs of HCMV DNA polymerase (
M54
) or helicase (
M105
) was protective against virus replication in the spleen following systemic challenge, with the protection level elicited by the
M54
DNA being comparable to that of DNA expressing the immunodominant
IE1
(pp89). Intracellular gamma interferon staining of CD8 T cells from mice immunized with either the
M54
or
M105
DNAs showed strong primary responses that recalled rapidly after viral challenge. M54- and M105-specific CD8 T cells were detected after the primary MCMV infection, but their levels were not consistently above the background level. The conserved, essential proteins of the CMVs thus represent a novel class of CD8 T-cell targets that may contribute to a successful HCMV vaccine strategy.
Lethal Silver-Haired Bat Rabies Virus Infection Can Be Prevented by Opening the Blood-Brain Barrier
Authors: Roy Anirban , Hooper D. Craig
Published: May 2007

Silver-haired bat rabies virus (SHBRV) infection induces a strong virus-specific immune response in the periphery of the host, but death is common due to the failure to open the blood-brain barrier (BBB) and deliver immune effectors to central nervous system (CNS) tissues. Mice with an SJL background are less susceptible to lethal infection with rabies viruses. In addition, these animals are known to have reduced hypothalamus-pituitary-adrenal (HPA) axis activity and an elevated capacity to mediate CNS inflammatory responses. We show here that approximately one-half of PLSJL mice survive an SHBRV infection that is invariably lethal for 129/SvEv mice. This difference is associated with the elevated capacity of PLSJL mice to mediate BBB permeability changes in response to the infection. The induction of more extensive BBB permeability and CNS inflammation in these animals results in greater virus clearance and improved survival. On the other hand, treatment of SHBRV-infected PLSJL mice with the steroid hormone dehydroepiandrosterone reduced BBB permeability changes and caused greater mortality. We conclude that the infiltration of immune effectors across the BBB is critical to surviving a rabies virus infection and that HPA axis activity may influence this process.
Selection of Mutations in the Connection and RNase H Domains of Human Immunodeficiency Virus Type 1 Reverse Transcriptase That Increase Resistance to 3′-Azido-3′-Dideoxythymidine
Authors: Brehm Jessica H. , Koontz Dianna , Meteer Jeffrey D. , Pathak Vinay , Sluis-Cremer Nicolas , Mellors John W.
Published: May 2007

Recent work indicates that mutations in the C-terminal domains of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) increase 3′-azido-3′-dideoxythymidine (AZT) resistance. Because it is not known whether AZT selects for mutations outside of the polymerase domain of RT, we carried out in vitro experiments in which HIV-1LAIor AZT-resistant HIV-1LAI(M41L/L210W/T215Y) was passaged in MT-2 cells in increasing concentrations of AZT. The first resistance mutations to appear in HIV-1LAIwere two polymerase domain thymidine analog mutations (TAMs), D67N and K70R, and two novel mutations, A371V in the connection domain and Q509L in the RNase H domain, that together conferred up to 90-fold AZT resistance. Thereafter, the T215I mutation appeared but was later replaced by T215F, resulting in a large increase in AZT resistance (∼16,000-fold). Mutations in the connection and RNase H domains were not selected starting with AZT-resistant virus (M41L/L210W/T215Y). The roles of A371V and Q509L in AZT resistance were confirmed by site-directed mutagenesis: A371V and Q509L together increased AZT resistance ∼10- to 50-fold in combination with TAMs (M41L/L210W/T215Y or D67N/K70R/T215F) but had a minimal effect without TAMs (1.7-fold). A371V and Q509L also increased cross-resistance with TAMs to lamivudine and abacavir, but not stavudine or didanosine. These results provide the first evidence that mutations in the connection and RNase H domains of RT can be selected in vitro by AZT and confer greater AZT resistance and cross-resistance to nucleoside RT inhibitors in combination with TAMs in the polymerase domain.
Thermolabilizing Pseudoreversions in Reovirus Outer-Capsid Protein μ1 Rescue the Entry Defect Conferred by a Thermostabilizing Mutation
Authors: Agosto Melina A. , Middleton Jason K. , Freimont Elaine C. , Yin John , Nibert Max L.
Published: May 2007

Heat-resistant mutants selected from infectious subvirion particles of mammalian reoviruses have determinative mutations in the major outer-capsid protein μ1. Here we report the isolation and characterization of intragenic pseudoreversions of one such thermostabilizing mutation. From a plaque that had survived heat selection, a number of viruses with one shared mutation but different second-site mutations were isolated. The effect of the shared mutation alone or in combination with second-site mutations was examined using recoating genetics. The shared mutation, D371A, was found to confer (i) substantial thermostability, (ii) an infectivity defect that followed attachment but preceded viral protein synthesis, and (iii) resistance to μ1 rearrangement in vitro, with an associated failure to lyse red blood cells. Three different second-site mutations were individually tested in combination with D371A and found to wholly or partially revert these phenotypes. Furthermore, when tested alone in recoated particles, each of these three second-site mutations conferred demonstrable thermolability. This and other evidence suggest that pseudoreversion of μ1-based thermostabilization can occur by a general mechanism of μ1-based thermolabilization, not requiring a specific compensatory mutation. The thermostabilizing mutation D371A as well as 9 of the 10 identified second-site mutations are located near contact regions between μ1 trimers in the reovirus outer capsid. The availability of both thermostabilizing and thermolabilizing mutations in μ1 should aid in defining the conformational rearrangements and mechanisms involved in membrane penetration during cell entry by this structurally complex nonenveloped animal virus.
Roles of Phosphatidylinositol 3-Kinase and NF-κB in Human Cytomegalovirus-Mediated Monocyte Diapedesis and Adhesion: Strategy for Viral Persistence
Authors: Smith M. Shane , Bivins-Smith Elizabeth R. , Tilley A. Michael , Bentz Gretchen L. , Chan Gary , Minard Jessica , Yurochko Andrew D.
Published: May 2007

Infected peripheral blood monocytes are proposed to play a key role in the hematogenous dissemination of human cytomegalovirus (HCMV) to tissues, a critical step in the establishment of HCMV persistence and the development of HCMV-associated diseases. We recently provided evidence for a unique strategy involved in viral dissemination: HCMV infection of primary human monocytes promotes their transendothelial migration and differentiation into proinflammatory macrophages permissive for the replication of the original input virus. To decipher the mechanism of hematogenous spread, we focused on the viral dysregulation of early cellular processes involved in transendothelial migration. Here, we present evidence that both phosphatidylinositol 3-kinase [PI(3)K] and NF-κB activities were crucial for the HCMV induction of monocyte motility and firm adhesion to endothelial cells. We found that the β1integrins, the β2integrins, intracellular adhesion molecule 1 (ICAM-1), and ICAM-3 were upregulated following HCMV infection and that they played a key role in the firm adhesion of infected monocytes to the endothelium. The viral regulation of adhesion molecule expression is complex, with PI(3)K and NF-κB affecting the expression of each adhesion molecule at different stages of the expression cascade. Our data demonstrate key roles for PI(3)K and NF-κB signaling in the HCMV-induced cellular changes in monocytes and identify the biological rationale for the activation of these pathways in infected monocytes, which together suggest a mechanism for how HCMV promotes viral spread to and persistence within host organs.