bluuebunny/crossref_metadata_2025 huggingface dataset:
ConformationalEpitopes Recognized by Protective Anti-Neisserial Surface Protein AAntibodies
Authors: Hou Victor C. , Moe Gregory R. , Raad Zyde , Wuorimaa Tomi , Granoff Dan M.
Published: November 2003

NspA
is a conserved membrane protein that elicits protective antibody
responses in mice against
Neisseria meningitidis
. A recent
crystallographic study showed that NspA adopts an eight-strandedβ
-barrel structure when reconstituted in detergent. In order to
define the segments of NspA-containing epitopes recognized by
protective murine anti-NspA antibodies, we studied the binding of two
bactericidal and protective anti-NspA monoclonal antibodies (MAbs),
AL12 and 14C7. Neither MAb binds to overlapping synthetic peptides
(10-mers, 12-mers, and cyclic 12-mers) corresponding to the entire
mature sequence of NspA, or to denatured recombinant NspA (rNspA),
although binding to the protein can be restored by refolding in
liposomes. Based on the ability of the two MAbs to bind to
Escherichia coli
microvesicles prepared from a set of rNspA
variants created by site-specific mutagenesis, the most important
contacts between the MAbs and NspA appear to be located within the LGG
segment of loop 3. The conformation of loop 2 also appears to be an
important determinant, as particular combinations of residues in this
segment resulted in loss of antibody binding. Thus, the two anti-NspA
MAbs recognize discontinuous conformational epitopes that result from
the close proximity of loops 2 and 3 in the three-dimensional structure
of NspA. The data suggest that optimally immunogenic vaccines using
rNspA will require formulations that permit proper folding of the
protein.
TheMycobacterium tuberculosisComplex-Restricted Genecfp32Encodes an Expressed Protein That Is Detectable inTuberculosis Patients and Is Positively Correlated with PulmonaryInterleukin-10
Authors: Huard Richard C. , Chitale Sadhana , Leung Mary , Lazzarini Luiz Claudio Oliveira , Zhu Hongxia , Shashkina Elena , Laal Suman , Conde Marcus B. , Kritski AfrÂnio L. , Belisle John T. , Kreiswirth Barry N. , Lapa e Silva José Roberto , Ho John L.
Published: November 2003

Human tuberculosis (TB) is caused by the bacillusMycobacteriumtuberculosis, a subspecies of theM.tuberculosiscomplex (MTC) of mycobacteria. Postgenomic dissection of theM. tuberculosisproteome is ongoing and critical to furthering our understanding of factors mediatingM. tuberculosispathobiology. Towards this end, a 32-kDa putative glyoxalase in the culture filtrate (CF) of growingM. tuberculosis(originally annotated as Rv0577 and hereafter designated CFP32) was identified, cloned, and characterized. Thecfp32gene is MTC restricted, and the gene product is expressed ex vivo as determined by the respective Southern and Western blot testing of an assortment of mycobacteria. Moreover, thecfp32gene sequence is conserved within the MTC, as no polymorphisms were found in the testedcfp32PCR products upon sequence analysis. Western blotting ofM.tuberculosissubcellular fractions localized CFP32 predominantly to the CF and cytosolic compartments. Data to support the in vivo expression of CFP32 were provided by the serum recognition of recombinant CFP32 in 32% of TB patients by enzyme-linked immunosorbent assay (ELISA) as well as the direct detection of CFP32 by ELISA in the induced sputum samples from 56% of pulmonary TB patients. Of greatest interest was the observation that, per sample, sputum CFP32 levels (a potential indicator of increasing bacterial burden) correlated with levels of expression in sputum of interleukin-10 (an immunosuppressive cytokine and a putative contributing factor to disease progression) but not levels of gamma interferon (a key cytokine in the protective immune response in TB), as measured by ELISA. Combined, these data suggest that CFP32 serves a necessary biological function(s) in tubercle bacilli and may contribute to theM. tuberculosispathogenic mechanism. Overall, CFP32 is an attractive target for drug and vaccine design as well as new diagnostic strategies.
Identification and Characterization ofpptA: a Gene Involved in the Phase-Variable Expression ofPhosphorylcholine on Pili ofNeisseriameningitidis
Authors: Warren Matthew J. , Jennings Michael P.
Published: November 2003

Pili of pathogenicNeisseriaare major virulence factors associated with adhesion, cytotoxicity, twitching motility, autoaggregation, and DNA transformation. Pili are modified posttranslationally by the addition of phosphorylcholine. However, no genes involved in either the biosynthesis or the transfer of phosphorylcholine inNeisseria meningitidishave been identified. In this study, we identified five candidate open reading frames (ORFs) potentially involved in the biosynthesis or transfer of phosphorylcholine to pilin inN. meningitidis. Insertional mutants were constructed for each ORF inN.meningitidisstrain C311#3 to determine their effect on phosphorylcholine expression. The effect of the mutant ORFs on the modification by phosphorylcholine was analyzed by Western analysis with phosphorylcholine-specific monoclonal antibody TEPC-15. Analysis of the mutants showed that ORF NMB0415, now defined aspptA(pilin phosphorylcholine transferase A), is involved in the addition of phosphorylcholine to pilin inN. meningitidis. Additionally, the phase variation (high frequency on-off switching of expression) of phosphorylcholine on pilin is due to changes in a homopolymeric guanosine tract inpptA.
Heterologous Priming-Boosting Immunization of Cattle withMycobacterium tuberculosis85A Induces Antigen-Specific T-Cell Responses
Authors: Taracha Evans L. N. , Bishop Richard , Musoke Antony J. , Hill Adrian V. S. , Gilbert Sarah C.
Published: November 2003

Heterologous priming-boosting vaccination regimens involving priming with plasmid DNA antigen constructs and inoculating (boosting) with the same recombinant antigen expressed in replication-attenuated poxviruses have recently been demonstrated to induce immunity, based on CD4+- and CD8+-T-cell responses, against several diseases in both rodents and primates. We show that similar priming-boosting vaccination strategies using the 85A antigen ofMycobacterium tuberculosisare effective in inducing antigen-specific gamma interferon-secreting CD4+and CD8+T cells, detected by a bovine enzyme-linked immunospot assay, inBos indicuscattle. T-cell responses induced by priming with either plasmid DNA or fowlpox virus 85A constructs were enhanced by boosting with modified vaccinia virus Ankara expressing the same antigen administered intradermally. On the basis of the data, it appears that intradermal priming was more effective than intramuscular delivery of the priming dose for boosting with the modified vaccinia virus Ankara strain in cattle. Using either fowlpox virus or DNA priming, there was a significant bias toward induction of CD4+- rather than CD8+-T-cell responses. These data illustrate the general applicability of priming-boosting vaccination strategies for induction of antigen-specific T-cell responses and suggest that the method may be useful for development of veterinary vaccines.
Mitogenic Effect ofBartonella bacilliformison Human Vascular Endothelial Cells and Involvement of GroEL
Authors: Minnick Michael F. , Smitherman Laura S. , Samuels D. Scott
Published: November 2003

Bartonellae are bacterial pathogens for a wide variety of mammals. In humans, bartonellosis can result in angioproliferative lesions that are potentially life threatening to the patient, including bacillary angiomatosis, bacillary peliosis, and verruga peruana. The results of this study show thatBartonella bacilliformis, the agent of Oroya fever and verruga peruana, produces a proteinaceous mitogen for human vascular endothelial cells (HUVECs) that acts in a dose-dependent fashion in vitro with maximal activity at ≥72 h of exposure and results in a 6- to 20-fold increase in cell numbers relative to controls. The mitogen increases bromodeoxyuridine (BrdU) incorporation into HUVECs by almost twofold relative to controls. The mitogen is sensitive to heat and trypsin but is not affected by the lipopolysaccharide inhibitor polymyxin B. The mitogen does not affect caspase 3 activity in HUVECs undergoing serum starvation-induced apoptosis. TheBartonellamitogen was found in bacterial culture supernatants, the soluble cell lysate fraction, and, to a lesser degree, in insoluble cell fractions of the bacterium. In contrast, soluble cell lysate fractions from closely relatedB. henselae, although possessing significant mitogenicity for HUVECs, resulted in only about a twofold increase in cell numbers. Biochemical and immunological analyses identified GroEL as a participant in the observed HUVEC mitogenicity. AB. bacilliformisstrain containing the intactgroES-groELoperon on a multicopy plasmid was generated and used to demonstrate a correlation between HUVEC mitogenicity and GroEL levels in the lysate (r2= 0.85). Antiserum to GroEL significantly inhibited mitogenicity of the lysate. Data also show that GroEL is located in the soluble and insoluble fractions (including inner and outer membranes) of the cell and is actively secreted byB. bacilliformis.
Synergism ofGamma Interferon and Interleukin-5 in the Control of MurineFilariasis
Authors: Saeftel Michael , Arndt Manuela , Specht Sabine , Volkmann Lars , Hoerauf Achim
Published: November 2003

There has been a prevailing perception that Th1 and Th2 immune responses induce antagonistic immune effector mechanisms during an infection. We investigated the role of the Th1 cytokine gamma interferon (IFN-γ) and the Th2 cytokine interleukin-5 (IL-5) in murine filariasis infections with the rodent filarial nematodeLitomosoides sigmodontiswith regard to immune responses to the parasite. Earlier data showed an important role for IL-5 and IFN-γ in effective immune responses to filarial infection. Therefore, in this study it was asked whether IL-5 and IFN-γ act synergistically or antagonistically. Indeed, IL-5 as well as IFN-γ knockout (KO) mice show a higher worm load than the wild-type controls. IFN-γ/IL-5 double-KO mice had a significantly higher worm load than any of the single-KO mice, suggesting a synergism between IFN-γ and IL-5 in controlling worm infection. Neutrophils are known to play an important role for the containment and encapsulation process of the worms. In infected IFN-γ KO, IL-5 KO, and IFN-γ/IL-5 double-KO mice, neutrophils were significantly reduced in chemotactic activity levels compared to controls. In addition, the level of phagocytosis activity of neutrophils from IFN-γ/IL-5 double-KO mice was further decreased in comparison to that of the single-KO mice. Levels of tumor necrosis factor alpha, which is an important factor for neutrophil activation, were found to be reduced in macrophages from KO mice. In conclusion, these results argue for immune effector mechanisms in murine filarial infection that are dependent on both IFN-γ and IL-5. Synergistic effects of the two cytokines may be mediated, at least in part, by neutrophils for the control of adult worms.
Removal of
Wolbachia
from
Brugia pahangi
Is Closely Linked to Worm Death and Fecundity but Does Not Result in Altered Lymphatic Lesion Formation in Mongolian Gerbils (
Meriones unguiculatus
)
Authors: Chirgwin Sharon R. , Coleman Sharon U. , Porthouse Kristina H. , Nowling Jena M. , Punkosdy George A. , Klei Thomas R.
Published: November 2003

Approximately 30 years ago, researchers reported intracellular bacteria in filarial nematodes. These bacteria are relatives of the arthropod symbiont
Wolbachia
and occur in many filarial nematodes, including
Brugia pahangi
and
Brugia malayi. Wolbachia
bacteria have been implicated in a variety of roles, including filaria development and fecundity and the pathogenesis of lymphatic lesions associated with filarial infections. However, the role of the bacteria in worm biology or filarial disease is still not clear. The present experiments support previous data showing that tetracycline eliminates or reduces
Wolbachia
bacteria in
B. pahangi
in vivo. The elimination of
Wolbachia
was closely linked to a reduction in female fecundity and the viability of both sexes, suggesting that the killing of
Wolbachia
is detrimental to
B. pahangi
. The gerbils treated with tetracycline showed reduced levels of interleukin-4 (IL-4) and IL-5 mRNA in renal lymph nodes and spleens compared with the levels in
B. pahangi
-infected gerbils not treated with tetracycline. However, similar findings were noted in
B. pahangi
-infected gerbils treated with ivermectin, suggesting that the loss of circulating microfilariae, not the reduction of
Wolbachia
bacteria, was associated with the altered cytokine profile. Despite the change in T-cell cytokines, there was no difference in the sizes of renal lymph nodes isolated from gerbils in each treatment group. Furthermore, the numbers, sizes, or cellular compositions of granulomas examined in the lymphatics or renal lymph nodes did not differ with treatment. These data suggest that
Wolbachia
may not play a primary role in the formation of lymphatic lesions in gerbils chronically infected with
B. pahangi
.
Effect ofMycobacterium bovisBCG Vaccinationon Mycobacterium-Specific Cellular Proliferation and TumorNecrosis Factor Alpha Production from Distinct Guinea PigLeukocytePopulations
Authors: Lasco Todd M. , Yamamoto Toshiko , Yoshimura Teizo , Allen Shannon Sedberry , Cassone Lynne , McMurray David N.
Published: November 2003

In this study, we focused on three leukocyte-rich guinea pig cell populations, bronchoalveolar lavage (BAL) cells, resident peritoneal cells (PC), and splenocytes (SPC). BAL cells, SPC, and PC were stimulated either with live attenuatedMycobacterium tuberculosisH37Ra or with live or heat-killed virulentM. tuberculosisH37Rv (multiplicity of infection of 1:100). Each cell population was determined to proliferate in response to heat-killed virulent H37Rv, whereas no measurable proliferative response could be detected upon stimulation with live mycobacteria. Additionally, this proliferative capacity (in SPC and PC populations) was significantly enhanced upon prior vaccination withMycobacterium bovisBCG. Accordingly, in a parallel set of experiments we found a strong positive correlation between production of antigen-specific bioactive tumor necrosis factor alpha (TNF-α) and prior vaccination with BCG. A nonspecific stimulus, lipopolysaccharide, failed to induce this effect on BAL cells, SPC, and PC. These results showed that production of bioactive TNF-α from mycobacterium-stimulated guinea pig cell cultures positively correlates with the vaccination status of the host and with the virulence of the mycobacterial strain.
Characterization of an Extracellular Virulence FactorMade by Group A
Streptococcus
with Homology to the
Listeria monocytogenes
Internalin Family ofProteins
Authors: Reid Sean D. , Montgomery Alison G. , Voyich Jovanka M. , DeLeo Frank R. , Lei Benfang , Ireland Robin M. , Green Nicole M. , Liu Mengyao , Lukomski Slawomir , Musser James M.
Published: November 2003

Leucine-rich
repeats (LRR) characterize a diverse array of proteins and function to
provide a versatile framework for protein-protein interactions.
Importantly, each of the bacterial LRR proteins that have been well
described, including those of
Listeria monocytogenes
,
Yersinia pestis
, and
Shigella flexneri
, have been
implicated in virulence. Here we describe an 87.4-kDa group A
Streptococcus
(GAS) protein (designated Slr, for streptococcal
leucine-rich) containing 10 1/2 sequential units of a 22-amino-acid
C-terminal LRR homologous to the LRR of the
L. monocytogenes
internalin family of proteins. In addition to the LRR domain,
slr
encodes a gram-positive signal secretion sequence
characteristic of a lipoprotein and a putative N-terminal domain with a
repeated histidine triad motif (HxxHxH). Real-time reverse
transcriptase PCR assays indicated that
slr
is transcribed
abundantly in vitro in the exponential phase of growth. Flow cytometry
confirmed that Slr was attached to the GAS cell surface. Western
immunoblot analysis of sera obtained from 80 patients with invasive
infections, noninvasive soft tissue infections, pharyngitis, and
rheumatic fever indicated that Slr is produced in vivo. An isogenic
mutant strain lacking
slr
was significantly less virulent in
an intraperitoneal mouse model of GAS infection and was significantly
more susceptible to phagocytosis by human polymorphonuclear leukocytes.
These studies characterize the first GAS LRR protein as an
extracellular virulence factor that contributes to pathogenesis and may
participate in evasion of the innate host
defense.
β-1,2- and α-1,2-LinkedOligomannosides Mediate Adherence of
Candida albicans
Blastospores to Human Enterocytes InVitro
Authors: Dalle Fredéric , Jouault Thierry , Trinel Pierre André , Esnault Jacques , Mallet Jean Maurice , d'Athis Philippe , Poulain Daniel , Bonnin Alain
Published: November 2003

Candida
albicans
is a commensal dimorphic yeast of the digestive tract
that causes hematogenously disseminated infections in immunocompromised
individuals. Endogenous invasive candidiasis develops from
C.
albicans
adhering to the intestinal epithelium. Adherence is
mediated by the cell wall surface, a domain composed essentially of
mannopyranosyl residues bound to proteins, the N-linked moiety of which
comprises sequences of α-1,2- and β-1,2-linked mannose
residues. β-1,2-linked mannosides are also associated with a
glycolipid, phospholipomannan, at the
C. albicans
surface. In
order to determine the roles of β-1,2 and α-1,2
oligomannosides in the
C. albicans
-enterocyte interaction, we
developed a model of adhesion of
C. albicans
VW32 blastospores
to the apical regions of differentiated Caco-2 cells. Preincubation of
yeasts with monoclonal antibodies (MAbs) specific for α-1,2 andβ
-1,2 mannan epitopes resulted in a dose-dependent decrease in
adhesion (50% of the control with a 60-μg/ml MAb
concentration). In competitive assays β-1,2 and α-1,2
tetramannosides were the most potent carbohydrate inhibitors, with
50% inhibitory concentrations of 2.58 and 6.99 mM, respectively.
Immunolocalization on infected monolayers with MAbs specific forα
-1,2 and β-1,2 oligomannosides showed that these
epitopes were shed from the yeast to the enterocyte surface. Taken
together, our data indicate that α-1,2 and β-1,2
oligomannosides are involved in the
C. albicans
-enterocyte
interaction and participate in the adhesion of the yeasts to the
mucosal
surface.
Phenotypic Switching and Mating Type Switching of
Candida glabrata
at Sites ofColonization
Authors: Brockert Paula J. , Lachke Salil A. , Srikantha Thyagarajan , Pujol Claude , Galask Rudolph , Soll David R.
Published: November 2003

Candida
glabrata
switches spontaneously at high frequency among the
following four graded phenotypes discriminated on agar containing 1 mM
CuSO
4
: white, light brown, dark brown (DB), and very dark
brown.
C. glabrata
also contains three mating type loci with a
configuration similar to that of the
Saccharomyces cerevisiae
mating type cassette system, suggesting it may also undergo cassette
switching at the expression locus
MTL1
. To analyze both
reversible, high-frequency phenotypic switching and mating type
switching at sites of colonization, primary samples from the oral
cavities and vaginal canals of three patients suffering from
C.
glabrata
vaginitis were clonally plated on agar containing
CuSO
4
. It was demonstrated that (i) in each vaginitis
patient, there was only one colonizing strain; (ii) an individual could
have vaginal colonization without oral colonization; (iii) phenotypic
switching occurred at sites of colonization; (iv) the DB phenotype
predominated at the site of infection in all three patients; (v)
genetically unrelated strains switched in similar, but not identical,
fashions and caused vaginal infection; (vi) different switch phenotypes
of the same strain could simultaneously dominate different body
locations in the same host; (vii) pathogenesis could be caused by cells
in different mating type classes; and (viii) mating type switching
demonstrated at both the genetic and transcription levels occurred in
one
host.
Recruitment of Complement Factor H-Like Protein 1 Promotes Intracellular Invasion by Group A Streptococci
Authors: Pandiripally Vinod , Wei Lin , Skerka Christine , Zipfel Peter F. , Cue David
Published: November 2003

Numerous microbial pathogens exploit complement regulatory proteins such as factor H (FH) and factor H-like protein 1 (FHL-1) for immune evasion. Fba is an FHL-1 and FH binding protein expressed on the surface of the human pathogenic bacterium,Streptococcus pyogenes, a common agent of pharyngeal, skin, and soft-tissue infections. In the present study, we demonstrate that Fba and FHL-1 work in concert to promote invasion of epithelial cells byS. pyogenes. Fba fragments were expressed as recombinant proteins and assayed for binding of FHL-1 and FH by Western blotting, enzyme-linked immunosorbent assay, and surface plasmon resonance. A binding site for FHL-1 and FH was localized to the N-terminal half of Fba, a region predicted to contain a coiled-coil domain. Deletion of this coiled-coil domain greatly reduced FHL-1 and FH binding. PepSpot analyses identified a 16-amino-acid segment of Fba which overlaps the coiled-coil domain that binds both FHL-1 and FH. To localize the Fba binding site in FHL-1 and FH, surface plasmon resonance was used to assess the interactions between the streptococcal protein and a series of recombinant FH deletion constructs. The Fba binding site was localized to short consensus repeat 7 (SCR 7), a domain common to FHL-1 and FH. SCR 7 contains a heparin binding site, and heparin was found to inhibit FHL-1 binding to Fba. FHL-1 promoted entry of Fba+group A streptococci into epithelial cells in a dose-dependent manner but did not affect invasion by an isogenicfbamutant. To our knowledge, this is the first report of a bacterial pathogen exploiting a soluble complement regulatory protein for entry into host cells.
Comparative Analysis of
Salmonella enterica
Serovar Typhimurium Biofilm Formation on Gallstones and on Glass
Authors: Prouty A. M. , Gunn J. S.
Published: November 2003

In this study, the roles of global regulators, motility, lipopolysaccharide, and exopolysaccharides were further characterized with respect to biofilm formation on both gallstones and glass surfaces. These studies show the complex nature of biofilms and demonstrate that characteristics observed for each biofilm are unique to the particular culture condition.
Interleukin-10 and Pathogenesis of Murine Ocular Toxoplasmosis
Authors: Lu Fangli , Huang Shiguang , Kasper Lloyd H.
Published: November 2003

To understand the role of interleukin-10 (IL-10) in ocular toxoplasmosis, we compared C57BL/6 (B6) and BALB/c background mice lacking a functional IL-10 gene (IL-10−/−) and B6 transgenic mice expressing IL-10 under the control of the IL-2 promoter. Increased cellular infiltration and necrosis were observed in the eye tissue of IL-10−/−mice of both the B6 and BALB/c backgrounds with associated changes in the levels of cytokines in serum. In contrast, there was no evidence of necrosis in the eye tissue from IL-10 transgenic mice following parasite exposure. Our results demonstrate that IL-10 is important in the regulation of inflammation during acute ocular toxoplasmosis.
Role of Urease Enzymes in Stability of a 10-Species Oral Biofilm Consortium Cultivated in a Constant-Depth Film Fermenter
Authors: Shu Man , Browngardt Christopher M. , Chen Yi-Ywan M. , Burne Robert A.
Published: November 2003

Using a 10-species oral biofilm consortium and defined mutants, we show that high-level capacity to generate ammonia from a common salivary substrate is needed to maintain community diversity. This model appears to be suitable for the study of the effects of individual genetic determinants on the ecology of oral biofilms.
The Activation of Bovine Plasminogen by PauA Is Not Required for Virulence ofStreptococcus uberis
Authors: Ward Philip N. , Field Terence R. , Rapier Christopher D. , Leigh James A.
Published: November 2003

A mutant ofStreptococcus uberiscarrying a single copy of ISS1withinpauAwas unable to activate bovine plasminogen. Contrary to a hypothesis postulated previously, this mutation did not alter the ability of the bacterium to grow in milk or to infect the lactating bovine mammary gland.
Lipopolysaccharidefrom the Periodontal PathogenPorphyromonas gingivalisPrevents Apoptosis of HL60-Derived Neutrophils InVitro
Authors: Murray D. A , Wilton J. M. A.
Published: November 2003

Lipopolysaccharide (LPS) fromPorphyromonas gingivalisprevented apoptosis of HL60-derived neutrophils, which could not be restored upon the addition of interleukin-10. Signaling ofP. gingivalisLPS through Toll-like receptor 2 (TLR2), not TLR4, may account for the inhibiting effect ofP. gingivalisLPS on apoptosis and provide a mechanism for the development of destructive periodontal inflammation.
Antibody Prophylaxis and Therapy against West NileVirus Infection in Wild-Type and ImmunodeficientMice
Authors: Engle Michael J. , Diamond Michael S.
Published: November 2003

West Nile virus (WNV) is a mosquito-borneFlavivirusthat causes encephalitis in a subset of susceptible humans. Current treatment for WNV infections is supportive, and no specific therapy or vaccine is available. In this study, we directly tested the prophylactic and therapeutic efficacy of polyclonal antibodies against WNV. Passive administration of human gamma globulin or mouse serum prior to WNV infection protected congenic wild-type, B-cell-deficient (μMT), and T- and B-cell-deficient (RAG1) C57BL/6J mice. Notably, no increased mortality due to immune enhancement was observed. Although immune antibody completely prevented morbidity and mortality in wild-type mice, its effect was not durable in immunocompromised mice: many μMT andRAG1mice eventually succumbed to infection. Thus, antibody by itself did not completely eliminate viral reservoirs in host tissues, consistent with an intact cellular immune response being required for viral clearance. In therapeutic postexposure studies, human gamma globulin partially protected against WNV-induced mortality. In μMT mice, therapy had to be initiated within 2 days of infection to gain a survival benefit, whereas in the wild-type mice, therapy even 5 days after infection reduced mortality. This time point is significant because between days 4 and 5, WNV was detected in the brains of infected mice. Thus, passive transfer of immune antibody improves clinical outcome even after WNV has disseminated into the central nervous system.
Effects of a Single Amino Acid Substitution within thep2 Region of Human Immunodeficiency Virus Type 1 on Packagingof Spliced ViralRNA
Authors: Russell Rodney S. , Roldan Ariel , Detorio Mervi , Hu Jing , Wainberg Mark A. , Liang Chen
Published: November 2003

Human
immunodeficiency virus type 1 encapsidates two copies of viral genomic
RNA in the form of a dimer. The dimerization process initiates via a
6-nucleotide palindrome that constitutes the loop of a viral RNA
stem-loop structure (i.e., stem loop 1 [SL1], also termed the
dimerization initiation site [DIS]) located within the
5′ untranslated region of the viral genome. We have now shown
that deletion of the entire DIS sequence virtually eliminated viral
replication but that this impairment was overcome by four second-site
mutations located within the matrix (MA), capsid (CA), p2, and
nucleocapsid (NC) regions of Gag. Interestingly, defective viral RNA
dimerization caused by the ΔDIS deletion was not significantly
corrected by these compensatory mutations, which did, however, allow
the mutated viruses to package wild-type levels of this DIS-deleted
viral RNA while excluding spliced viral RNA from encapsidation. Further
studies demonstrated that the compensatory mutation T12I located within
p2, termed MP2, sufficed to prevent spliced viral RNA from being
packaged into the ΔDIS virus. Consistently, theΔ
DIS-MP2 virus displayed significantly higher levels of
infectiousness than did the ΔDIS virus. The importance of
position T12 in p2 was further demonstrated by the identification of
four point mutations,T12D, T12E, T12G, and T12P, that resulted in
encapsidation of spliced viral RNA at significant levels. Taken
together, our data demonstrate that selective packaging of viral
genomic RNA is influenced by the MP2 mutation and that this represents
a major mechanism for rescue of viruses containing the ΔDIS
deletion.
Role of Nonstructural Proteins 3A and 3B in Host Rangeand Pathogenicity of Foot-and-Mouth DiseaseVirus
Authors: Pacheco Juan M. , Henry Tina M. , O'Donnell Vivian K. , Gregory Jason B. , Mason Peter W.
Published: November 2003

The
genome of foot-and-mouth disease virus (FMDV) differs from that of
other picornaviruses in that it encodes a larger 3A protein
(>50% longer than poliovirus 3A), as well as three
copies of protein 3B (also known as VPg). Previous studies have shown
that a deletion of amino acids 93 to 102 of the 153-codon 3A protein is
associated with an inability of a Taiwanese strain of FMDV (O/TAW/97)
to cause disease in bovines. Recently, an Asian virus with a second 3A
deletion (amino acids 133 to 143) has also been detected (N.
J. Knowles et al., J. Virol.
75:
1551-1556, 2001).
Genetically engineered viruses harboring the amino acids 93 to 102 or
133 to 143 grew well in porcine cells but replicated poorly in bovine
cells, whereas a genetically engineered derivative of the O/TAW/97
virus expressing a full-length 3A (strain A12) grew well in both cell
types. Interestingly, a virus with a deletion spanning amino acid 93 to
144 also grew well in porcine cells and caused disease in swine.
Further, genetically engineered viruses containing only a single copy
of VPg were readily recovered with the full-length 3A, the deleted 3A
(amino acids 93 to 102), or the “super” deleted forms
of 3A (missing amino acids 93 to 144). All of the single-VPg viruses
were attenuated in porcine cells and replicated poorly in bovine cells.
The single-VPg viruses produced a mild disease in swine, indicating
that the VPg copy number is an important determinant of host range and
virulence. The association of VPg copy number with increased virulence
in vivo may help to explain why all naturally occurring FMDVs have
retained three copies of
VPg.
Reduced Cell Turnover in Bovine LeukemiaVirus-Infected, Persistently LymphocytoticCattle
Authors: Debacq Christophe , Asquith Becca , Reichert Michal , Burny Arsène , Kettmann Richard , Willems Luc
Published: November 2003

Although
nucleotide analogs like bromodeoxyuridine have been extensively used to
estimate cell proliferation in vivo, precise dynamic parameters are
scarce essentially because of the lack of adequate mathematical models.
Besides recent developments on T cell dynamics, the turnover rates of B
lymphocytes are largely unknown particularly in the context of a
virally induced pathological disorder. Here, we aim to resolve this
issue by determining the rates of cell proliferation and death during
the chronic stage of the bovine leukemia virus (BLV) infection, called
bovine persistent lymphocytosis (PL). Our methodology is based on
direct intravenous injection of bromodeoxyuridine in association with
subsequent flow cytometry. By this in vivo approach, we show that the
death rate of PL B lymphocytes is significantly reduced (average death
rate, 0.057 day
−1
versus 0.156
day
−1
in the asymptomatic controls). Concomitantly,
proliferation of the PL cells is also significantly restricted compared
to the controls (average proliferation rate, 0.0046
day
−1
versus 0.0085 day
−1
). We
conclude that bovine PL is characterized by a decreased cell turnover
resulting both from a reduction of cell death and an overall impairment
of proliferation. The cell dynamic parameters differ from those
measured in sheep, an experimental model for BLV infection. Finally,
cells expressing p24 major capsid protein ex vivo were not BrdU
positive, suggesting an immune selection against proliferating
virus-positive lymphocytes. Based on a comparative leukemia approach,
these observations might help to understand cell dynamics during other
lymphoproliferative disease such as chronic lymphocytic leukemia or
human T-cell lymphotropic virus-induced adult T-cell leukemia in
humans.
HumanImmunodeficiency Virus Type 1 Fitness Is a Determining Factor in ViralRebound and Set Point in ChronicInfection
Authors: Trkola Alexandra , Kuster Herbert , Leemann Christine , Ruprecht Claudia , Joos Beda , Telenti Amalio , Hirschel Bernhard , Weber Rainer , Bonhoeffer Sebastian , Günthard Huldrych F.
Published: November 2003

Human
immunodeficiency virus type 1 (HIV-1) isolates from 20
chronically infected patients who participated in a structured
treatment interruption (STI) trial were studied to determine whether
viral fitness influences reestablishment of viremia. Viruses derived
from individuals who spontaneously controlled viremia had significantly
lower in vitro replication capacities than viruses derived from
individuals that did not control viremia after interruption of
antiretroviral therapy (ART), and replication capacities
correlated with pre-ART and post-STI viral set points. Of
note, no clinically relevant improvement of viral loads upon STI
occurred. Virus isolates from controlling and noncontrolling patients
were indistinguishable in terms of coreceptor usage, genetic subtype,
and sensitivity to neutralizing antibodies. In contrast, viruses from
controlling patients exhibited increased sensitivity to inhibition by
chemokines. Sensitivity to inhibition by RANTES correlated strongly
with slower replication kinetics of the virus isolates, suggesting a
marked dependency of these virus isolates on high coreceptor densities
on the target cells. In summary, our data indicate that viral fitness
is a driving factor in determining the magnitude of viral rebound and
viral set point in chronic HIV-1 infection, and thus fitness should be
considered as a parameter influencing the outcome of therapeutic
intervention in chronic
infection.
Immunoprotective Activity and Safety of a RespiratorySyncytial Virus Vaccine: Mucosal Delivery of Fusion Glycoprotein with aCpG OligodeoxynucleotideAdjuvant
Authors: Prince Gregory A. , Mond James J. , Porter David D. , Yim Kevin C. , Lan Steve J. , Klinman Dennis M.
Published: November 2003

CpG oligodeoxynucleotides (ODN) were identified that stimulated immunoglobulin production and cell proliferation in cotton rat cells in vitro. Three of these ODN were used as a mucosal adjuvant in the noses of cotton rats immunized via this route with respiratory syncytial virus fusion (F) protein. The CpG ODN markedly increased the cotton rat humoral neutralizing-antibody response to respiratory syncytial virus. Such immunized animals had a marked reduction in the production of infectious virus after a live-virus challenge. Animals immunized with the combination of F protein and CpG developed enhanced pulmonary pathology consisting of alveolitis and interstitial pneumonitis after a live-virus challenge. Similar enhanced disease has been seen in cotton rats and children immunized with formalin-inactivated respiratory syncytial virus.
Sequestration of p53 in the Cytoplasm by AdenovirusType 12 E1B 55-Kilodalton Oncoprotein Is Required for Inhibition ofp53-MediatedApoptosis
Authors: Zhao Lisa Y. , Liao Daiqing
Published: November 2003

The
adenovirus E1B 55-kDa protein is a potent inhibitor of p53-mediated
transactivation and apoptosis. The proposed mechanisms include
tethering the E1B repression domain to p53-responsive promoters via
direct E1B-p53 interaction. Cytoplasmic sequestration of p53 by the
55-kDa protein would impose additional inhibition on p53-mediated
effects. To investigate further the role of cytoplasmic sequestration
of p53 in its inhibition by the E1B 55-kDa protein we systematically
examined domains in both the Ad12 55-kDa protein and p53 that underpin
their colocalization in the cytoplasmic body and show that the
N-terminal transactivation domain (TAD) of p53 is essential for
retaining p53 in the cytoplasmic body. Deletion of amino acids 11 to 27
or even point mutation L22Q/W23S abolished the localization of p53 to
the cytoplasmic body, whereas other parts of TAD and the C-terminal
domain of p53 are dispensable. This cytoplasmic body is distinct from
aggresome associated with overexpression of some proteins, since it
neither altered vimentin intermediate filaments nor associated with
centrosome or ubiquitin. Formation of this structure is sensitive to
mutation of the Ad12 55-kDa protein. Strikingly, mutation S476/477A
near the C terminus of the Ad12 55-kDa protein eliminated the formation
of the cytoplasmic body. The equivalent residues in the Ad5 55-kDa
protein were shown to be critical for its ability to inhibit p53.
Indeed, Ad12 55-kDa mutants that cannot form a cytoplasmic body can no
longer inhibit p53-mediated effects. Conversely, the Ad12 55-kDa
protein does not suppress p53 mutant L22Q/W23S-mediated apoptosis.
Finally, we show that E1B can still sequester p53 that contains the
mitochondrial import sequence, thereby potentially preventing the
localization of p53 to mitochondria. Thus, cytoplasmic sequestration of
p53 by the E1B 55-kDa protein plays an important role in restricting
p53
activities.
A Recombinant Influenza A Virus Expressing anRNA-Binding-Defective NS1 Protein Induces High Levels of BetaInterferon and Is Attenuated inMice
Authors: Donelan Nicola R. , Basler Christopher F. , García-Sastre Adolfo
Published: November 2003

Previously
we found that the amino-terminal region of the NS1 protein of influenza
A virus plays a key role in preventing the induction of beta interferon
(IFN-β) in virus-infected cells. This region is characterized
by its ability to bind to different RNA species, including
double-stranded RNA (dsRNA), a known potent inducer of IFNs. In order
to investigate whether the NS1 RNA-binding activity is required for its
IFN antagonist properties, we have generated a recombinant influenza A
virus which expresses a mutant NS1 protein defective in dsRNA binding.
For this purpose, we substituted alanines for two basic amino acids
within NS1 (R38 and K41) that were previously found to be required for
RNA binding. Cells infected with the resulting recombinant virus showed
increased IFN-β production, demonstrating that these two amino
acids play a critical role in the inhibition of IFN production by the
NS1 protein during viral infection. In addition, this virus grew to
lower titers than wild-type virus in MDCK cells, and it was attenuated
in mice. Interestingly, passaging in MDCK cells resulted in the
selection of a mutant virus containing a third mutation at amino acid
residue 42 of the NS1 protein (S42G). This mutation did not result in a
gain in dsRNA-binding activity by the NS1 protein, as measured by an in
vitro assay. Nevertheless, the NS1 R38AK41AS42G mutant virus was able
to replicate in MDCK cells to titers close to those of wild-type virus.
This mutant virus had intermediate virulence in mice, between those of
the wild-type and parental NS1 R38AK41A viruses. These results suggest
not only that the IFN antagonist properties of the NS1 protein depend
on its ability to bind dsRNA but also that they can be modulated by
amino acid residues not involved in RNA
binding.
Human Cytomegalovirus Transmission from the Uterus tothe Placenta Correlates with the Presence of Pathogenic Bacteria andMaternalImmunity
Authors: Pereira Lenore , Maidji Ekaterina , McDonagh Susan , Genbacev Olga , Fisher Susan
Published: November 2003

Prenatal cytomegalovirus infection may cause pregnancy complications such as intrauterine growth restriction and birth defects. How virus from the mother traverses the placenta is unknown. PCR analysis of biopsy specimens of the maternal-fetal interface revealed that DNA sequences from cytomegalovirus were commonly found with those of herpes simplex viruses and pathogenic bacteria. Cytomegalovirus DNA and infected cell proteins were found more often in the decidua than in the placenta, suggesting that the uterus functions as a reservoir for infection. In women with low neutralizing titers, cytomegalovirus replicated in diverse decidual cells and placental trophoblasts and capillaries. In women with intermediate to high neutralizing titers, decidual infection was suppressed and the placenta was spared. Overall, cytomegalovirus virions and maternal immunoglobulin G were detected in syncytiotrophoblasts, villus core macrophages, and dendritic cells. These results suggest that the outcome of cytomegalovirus infection depends on the presence of other pathogens and coordinated immune responses to viral replication at the maternal-fetal interface.
CD8+T Cells Mediate Recovery andImmunopathology in West Nile VirusEncephalitis
Authors: Wang Yang , Lobigs Mario , Lee Eva , Müllbacher Arno
Published: November 2003

C57BL/6J mice infected intravenously with the Sarafend strain of West Nile virus (WNV) develop a characteristic central nervous system (CNS) disease, including an acute inflammatory reaction. Dose response studies indicate two distinct kinetics of mortality. At high doses of infection (108PFU), direct infection of the brain occurred within 24 h, resulting in 100% mortality with a 6-day mean survival time (MST), and there was minimal destruction of neural tissue. A low dose (103PFU) of infection resulted in 27% mortality (MST, 11 days), and virus could be detected in the CNS 7 days postinfection (p.i.). Virus was present in the hypogastric lymph nodes and spleens at days 4 to 7 p.i. Histology of the brains revealed neuronal degeneration and inflammation within leptomeninges and brain parenchyma. Inflammatory cell infiltration was detectable in brains from day 4 p.i. onward in the high-dose group and from day 7 p.i. in the low-dose group, with the severity of infiltration increasing over time. The cellular infiltrates in brain consisted predominantly of CD8+, but not CD4+, T cells. CD8+T cells in the brain and the spleen expressed the activation markers CD69 early and expressed CD25 at later time points. CD8+T-cell-deficient mice infected with 103PFU of WNV showed increased mortalities but prolonged MST and early infection of the CNS compared to wild-type mice. Using high doses of virus in CD8-deficient mice leads to increased survival. These results provide evidence that CD8+T cells are involved in both recovery and immunopathology in WNV infection.
The δ Region of Outer-Capsid Proteinμ1 Undergoes Conformational Change and Release from ReovirusParticles during CellEntry
Authors: Chandran Kartik , Parker John S. L. , Ehrlich Marcelo , Kirchhausen Tomas , Nibert Max L.
Published: November 2003

Cell
entry by reoviruses requires a large, transcriptionally active
subvirion particle to gain access to the cytoplasm. The features of
this particle have been the subject of debate, but three primary
candidates—the infectious subvirion particle (ISVP), ISVP*, and
core particle forms—that differ in whether putative membrane
penetration protein μ1 and adhesin σ1 remain particle
bound have been identified. Experiments with antibody reagents in this
study yielded new information about the steps in particle disassembly
during cell entry. Monoclonal antibodies specific for the δ
region of μ1 provided evidence for a conformational change inμ
1 and for release of the δ proteolytic fragment from
entering particles. Antiserum raised against cores provided evidence
for entry-related changes in particle structure and identified entering
particles that largely lack the δ fragment inside cells.
Antibodies specific for σ1 showed that it is also largely shed
from entering particles. Limited coimmunostaining with markers for late
endosomes and lysosomes indicated the particles lacking δ andσ
1 did not localize to those subcellular compartments, and
other observations suggested that both the particles and free δ
were released into the cytoplasm. Essentially equivalent findings were
obtained with native ISVPs and highly infectious recoated particles
containing wild-type proteins. Poorly infectious recoated particles
containing a hyperstable mutant form of μ1, however, showed no
evidence for the in vitro and intracellular changes in particle
structure normally detected by antibodies, and these particles instead
accumulated in late endosomes or lysosomes. Recoated particles with
hyperstable μ1 were also ineffective at mediating erythrocyte
lysis in vitro and promoting α-sarcin coentry and intoxication
of cells in cultures. Based on these and other findings, we propose
that ISVP* is a transient intermediate in cell entry which mediates
membrane penetration and is then further uncoated in the cytoplasm to
yield particles, resembling cores, that largely lack the δ
fragment ofμ
1.
DetailedAnalysis of the CD8
+
T-Cell Response followingAdenovirusVaccination
Authors: Yang Teng Chih , Dayball Kelley , Wan Yong Hong , Bramson Jonathan
Published: November 2003

We
examined CD8
+
T-cell expansion and function
following intramuscular immunization with a recombinant adenovirus.
This study has identified a number of properties which may explain the
strong immunogenicity of adenovirus vectors: (i) the ability to deliver
large amounts of antigen into the lymphoid tissues, (ii) the ability to
induce rapid expansion and migration of CD8
+
T cells
throughout the lymphatics, and (iii) the ability to produce a
sustained, high-level CD8
+
T-cell
response.
Identificationof Proteins Associated with Murine Gammaherpesvirus68Virions
Authors: Bortz Eric , Whitelegge Julian P. , Jia Qingmei , Zhou Z. Hong , Stewart James P. , Wu Ting-Ting , Sun Ren
Published: November 2003

Murine
gammaherpesvirus 68 (MHV68 [also known asγ
HV-68]) is distinguished by its ability to
replicate to high titers in cultured cells, making it an excellent
candidate for studying gammaherpesvirus virion composition.
Extracellular MHV68 virions were isolated, and abundant
virion-associated proteins were identified by mass spectrometry. Five
nucleocapsid protein homologues, the tegument protein homologue encoded
by open reading frame (ORF) 75c, and envelope glycoproteins B and H
were detected. In addition, gene products from MHV68 ORF20,
ORF24, ORF28, ORF45, ORF48, and ORF52 were identified in
association with virions, suggesting that these gammaherpesvirus genes
are involved in the early phase of infection or virion assembly and
egress.
Adenovirus-Mediated Gene Transfer of Transforming Growth Factor-β
3
, but Not Transforming Growth Factor-β
1
, Inhibits Constrictive Remodeling and Reduces Luminal Loss After Coronary Angioplasty
Authors: Kingston Paul A. , Sinha Sanjay , Appleby Clare E. , David Anne , Verakis Thomas , Castro Maria G. , Lowenstein Pedro R. , Heagerty Anthony M.
Published: November 2003

Background—

Extracellular matrix (ECM) remodeling is central to the development of restenosis after PTCA. Substantial evidence implicates transforming growth factor-β
1
(TGF-β
1
), a regulator of ECM deposition by vascular cells, in its pathogenesis. TGF-β
3
reduces TGF-β
1
—induced ECM deposition in cutaneous wounds. We therefore investigated the effects of intracoronary expression of TGF-β
3
and TGF-β
1
on luminal loss after angioplasty.

Methods and Results—

Porcine coronary arteries received an adenovirus expressing TGF-β
3
, TGF-β
1
, or
lacZ
(β-galactosidase), or PBS only, at the site of angioplasty. Morphometric analysis 28 days after angioplasty confirmed reduced luminal loss in TGF-β
3
vessels (−0.65±0.10 mm
2
) compared with
lacZ
(−1.18±0.19 mm
2
) or PBS only (−1.19±0.17 mm
2
;
P
=0.003). Luminal loss was not reduced in TGF-β
1
vessels (−1.02±0.19 mm
2
;
P
=0.48). An increase in the external elastic lamina area in TGF-β
3
–treated vessels (+0.73±0.32 mm
2
) contrasted with decreases in control vessels (mean, −0.53±0.17 mm
2
;
P
=0.001) and TGF-β
1
vessels (−0.87±0.34 mm
2
;
P
=0.003). Collagen content increased at the site of injury in TGF-β
3
–treated vessels (26.1±14.2%) but decreased in the
lacZ
(−22.8±6.6%) and PBS-only (−23.4±7.0%;
P
=0.002) groups and was not significantly changed in TGF-β
1
–treated vessels.

Conclusions—

Expression of TGF-β
3
inhibits constrictive remodeling after PTCA and reduces luminal loss. This is accompanied by increased adventitial collagen, which may act as an external “scaffold” preventing vessel constriction. These findings confirm the potential of gene therapies that modify ECM remodeling for prophylaxis of restenosis.