bluuebunny/crossref_metadata_2025_split huggingface dataset:
Critical Roles for
stx
2
,
eae
, and
tir
in Enterohemorrhagic
Escherichia coli
-Induced Diarrhea and Intestinal Inflammation in Infant Rabbits
Authors: Ritchie Jennifer M. , Thorpe Cheleste M. , Rogers Arlin B. , Waldor Matthew K. 
Published: November 2003

ABSTRACT

Enterohemorrhagic
Escherichia coli
(EHEC) is a group of food-borne pathogens
that can cause diarrhea, colitis, and the hemolytic uremic syndrome
(HUS). The importance of several of the proposed EHEC virulence factors
lacks experimental verification in animal models. The limitations of
current animal models led us to reexamine the infant rabbit model for
the study of EHEC pathogenicity. Here, we report that intragastric
inoculation of a Shiga toxin 2 (Stx2)-producing
E. coli
O157:H7 clinical isolate into infant rabbits led to severe diarrhea and
intestinal inflammation but no signs of HUS. We constructed a set of
isogenic derivatives of this isolate with deletions in several putative
virulence genes, including
stx
2
,
eae
,
tir
, and
ehxA
, to investigate the contribution of
individual virulence factors to EHEC pathogenicity.
stx
2
increased the severity and duration of
EHEC-induced diarrhea. Furthermore, although
stx
2
had no role in EHEC intestinal colonization nor was it required for
EHEC-induced inflammation,
stx
2
altered how the
host responded to EHEC infection by promoting heterophilic infiltration
of the colonic epithelium and lamina propria. Intragastric inoculation
of purified Stx2 also induced inflammation and diarrhea in this model.
Diarrhea and intestinal inflammation were also dependent on EHEC
colonization, as EHEC derivatives with deletions in
eae
and
tir
did not colonize, form attaching and effacing lesions, or
develop clinical signs of disease. Our studies indicate that infant
rabbits are a useful model for investigation of the intestinal stage of
EHEC pathogenesis and suggest that Shiga toxin may play a critical role
in causing diarrhea and inflammation in patients infected with
EHEC.
PneumococcalSurface Protein A Is Expressed In Vivo, and Antibodies to PspA AreEffective for Therapy in a Murine Model of PneumococcalSepsis
Authors: Swiatlo E. , King J. , Nabors G. S. , Mathews B. , Briles D. E. 
Published: November 2003

ABSTRACT

Pneumococcal
surface protein A (PspA) is an immunogenic protein expressed on the
surface of all strains of
Streptococcus pneumoniae
(pneumococcus) and induces antibodies which protect against invasive
infection in mice. Pneumococci used for infectious challenge in
protection studies are typically collected from cultures grown in
semisynthetic medium in vitro. The purpose of these studies is to
confirm that PspA is expressed by pneumococci during growth in vivo at
a level sufficient for antibodies to PspA to be protective. Mice were
actively immunized with purified PspA or by passive transfer of
monoclonal antibody (MAb) and challenged with a capsular type 3 strain
in diluted whole blood from bacteremic mice. All were protected against
challenge with 10 times the 50% lethal dose (LD
50
),
and mice challenged with 1,000 times the LD
50
had increased
survival compared with controls. Additionally, nonimmune mice treated
with MAbs to PspA or PspA immune serum at 6 and 12 h after
infection with 10 times the LD
50
also showed increased
survival. Northern blot analysis of RNA from pneumococci grown either
in vitro or in vivo showed similar levels of PspA mRNA. These results
demonstrate that PspA is expressed in vivo in a mouse model and that
immunization with PspA induces antibodies to an antigen which is
expressed during the course of invasive infection.
Immunotherapy with antibodies to PspA may have some utility in treating
pneumococcal infections in
humans.
CD8+-T-CellResponses of Mycobacterium-Infected Mice to a Newly Identified MajorHistocompatibility Complex Class I-Restricted Epitope Shared byProteins of the ESAT-6Family
Authors: Majlessi Laleh , Rojas Marie-Jésus , Brodin Priscille , Leclerc Claude 
Published: November 2003

ABSTRACTHere we describe the identification of a new CD8+-T-cell epitope, the GYAGTLQSL nonamer, shared by the TB10.3 and TB10.4 proteins of theMycobacterium tuberculosisESAT-6 family. Cytotoxic T cells from mycobacterium-infected mice efficiently recognized this epitope. GYAGTLQSL-specific T-cell hybridomas, which were able to recognizeMycobacterium bovisBCG-infected macrophages, were generated and now allow investigation of mycobacterial-antigen processing through the major histocompatibility complex class I pathway.
Inducible Nitric Oxide Synthase Regulates Production of Isoprostanes In Vivo during Chlamydial Genital Infection in Mice
Authors: Ramsey K. H. , Sigar I. M. , Rana S. V. , Gupta J. , Holland S. M. , Byrne G. I. , Morrow J. D. 
Published: November 2003

ABSTRACTUrinary nitrite and F2-isoprostanes, an index of oxidant stress, were elevated during chlamydial genital infection of mice. Enhancement of urinary nitrite and F2-isoprostanes was observed in phagocyte oxidase-deficient mice. Inhibition of inducible nitric oxide synthase reduced isoprostane excretion. We conclude that nitrogen radicals induce F2-isoprostane production and excretion during murine chlamydial genital infection.
High Levels of Susceptibility and T Helper 2 Response in MyD88-Deficient Mice Infected withLeishmania majorAre Interleukin-4 Dependent
Authors: Debus Andrea , Gläsner Joachim , Röllinghoff Martin , Gessner André 
Published: November 2003

ABSTRACTMyeloid differentiation protein 88 (MyD88) is a general adaptor for the signaling cascade through receptors of the Toll/IL-1R family. When infected withLeishmania majorparasites, MyD88-deficient mice displayed a dramatically enhanced parasite burden in their tissues similar to that found in susceptible BALB/c mice. In contrast, MyD88 knockout mice did not develop ulcerating lesions despite a lack of interleukin-12 (IL-12) production and a predominant T helper 2 cell response. Blockade of IL-4 produced early (day 1) after infection restored a protective T helper 1 response in MyD88 knockout mice.
Targeting ofAdenovirus via Genetic Modification of the Viral Capsid Combined with aProteinBridge
Authors: Korokhov Nikolay , Mikheeva Galina , Krendelshchikov Alexander , Belousova Natalya , Simonenko Vera , Krendelshchikova Valentina , Pereboev Alexander , Kotov Alexander , Kotova Olga , Triozzi Pierre L. , Aldrich Wayne A. , Douglas Joanne T. , Lo Kin-Ming , Banerjee Papia T. , Gillies Stephen D. , Curiel David T. , Krasnykh Victor 
Published: November 2003

ABSTRACTA potential barrier to the development of genetically targeted adenovirus (Ad) vectors for cell-specific delivery of gene therapeutics lies in the fact that several types of targeting protein ligands require posttranslational modifications, such as the formation of disulfide bonds, which are not available to Ad capsid proteins due to their nuclear localization during assembly of the virion. To overcome this problem, we developed a new targeting strategy, which combines genetic modifications of the Ad capsid with a protein bridge approach, resulting in a vector-ligand targeting complex. The components of the complex associate by virtue of genetic modifications to both the Ad capsid and the targeting ligand. One component of this mechanism of association, the Fc-binding domain ofStaphylococcus aureusprotein A, is genetically incorporated into the Ad fiber protein. The ligand is comprised of a targeting component fused with the Fc domain of immunoglobulin, which serves as a docking moiety to bind to these genetically modified fibers during the formation of the Ad-ligand complex. The modular design of the ligand solves the problem of structural and biosynthetic compatibility with the Ad and thus facilitates targeting of the vector to a variety of cellular receptors. Our study shows that targeting ligands incorporating the Fc domain and either an anti-CD40 single-chain antibody or CD40L form stable complexes with protein A-modified Ad vectors, resulting in significant augmentation of gene delivery to CD40-positive target cells. Since this gene transfer is independent of the expression of the native Ad5 receptor by the target cells, this strategy results in the derivation of truly targeted Ad vectors suitable for tissue-specific gene therapy.
Role of the Cytoplasmic Domain of the NewcastleDisease Virus Fusion Protein in Association with LipidRafts
Authors: Dolganiuc V. , McGinnes L. , Luna E. J. , Morrison T. G. 
Published: November 2003

ABSTRACT

To
explore the association of the Newcastle disease virus (NDV) fusion (F)
protein with cholesterol-rich membrane domains, its localization in
detergent-resistant membranes (DRMs) in transfected cells was
characterized. After solubilization of cells expressing the F protein
with 1% Triton X-100 at 4°C, ca. 40% of total,
cell-associated F protein fractionated with classical DRMs with
densities of 1.07 to l.14 as defined by flotation into sucrose density
gradients. Association of the F protein with this cell fraction was
unaffected by the cleavage of F
0
to F
1
and
F
2
or by coexpression of the NDV attachment protein, the
hemagglutinin-neuraminidase protein (HN). Furthermore, elimination by
mutation, of potential palmitate addition sites in and near the
F-protein transmembrane domain had no effect on F-protein association
with DRMs. Rather, specific deletions of the cytoplasmic domain of the
F protein eliminated association with classical DRMs. Comparisons of
deletions that affected fusion activity of the protein and deletions
that affected DRM association suggested that there is no direct link
between the cell-cell fusion activity of the F protein and DRM
association. Furthermore, depletion of cholesterol from cells
expressing F and HN protein, while eliminating DRM association, had no
effect on the ability of these cells to fuse with avian red blood
cells. These results suggest that specific localization of the F
protein in cholesterol-rich membrane domains is not required for
cell-to-cell fusion. Paramyxovirus F-protein cytoplasmic domains have
been implicated in virus assembly. The results presented here raise the
possibility that the cytoplasmic domain is important in virus assembly
at least in part because it directs the protein to cholesterol-rich
membrane
domains.
Macrophage-Tropic Simian/Human Immunodeficiency VirusChimeras Use CXCR4, Not CCR5, for Infections of Rhesus MacaquePeripheral Blood Mononuclear Cells and AlveolarMacrophages
Authors: Igarashi Tatsuhiko , Donau Olivia K. , Imamichi Hiromi , Dumaurier Marie-Jeanne , Sadjadpour Reza , Plishka Ronald J. , Buckler-White Alicia , Buckler Charles , Suffredini Anthony F. , Lane H. Clifford , Moore John P. , Martin Malcolm A. 
Published: November 2003

ABSTRACTAfter the nearly complete and irreversible depletion of CD4+T lymphocytes induced by highly pathogenic simian/human immunodeficiency virus chimeric viruses (SHIVs) during infections of rhesus monkeys, tissue macrophages are able to sustain high levels (>106viral RNA copies/ml) of plasma viremia for several months. We recently reported that the virus present in the plasma during the late macrophage phase of infection had acquired changes that specifically targeted the V2 region of gp120 (H. Imamichi et al., Proc. Natl. Acad. Sci. USA99:13813-13818, 2002); some of these SHIV variants were macrophage-tropic (M-tropic). Those findings have been extended by examining the tropic properties, coreceptor usage, and gp120 structure of five independent SHIVs recovered directly from lymph nodes of late-stage animals. All of these tissue-derived SHIV isolates were able to infect alveolar macrophages. These M-tropic SHIVs used CXCR4, not CCR5, for infections of rhesus monkey PBMC and primary alveolar macrophages. Because the starting highly pathogenic T-tropic SHIV inoculum also utilized CXCR4, these results indicate that the acquisition of M-tropism in the SHIV-macaque system is not accompanied by a change in coreceptor usage. Compared to the initial T-tropic SHIV inoculum, tissue-derived M-tropic SHIVs from individual infected animals carry gp120s containing similar changes (specific amino acid deletions, substitutions, and loss of N-linked glycosylation sites), primarily within the V1 and/or V2 regions of gp120.
High-Frequency Homologous Recombination betweenBaculoviruses Involves DNAReplication
Authors: Kamita Shizuo George , Maeda Susumu , Hammock Bruce D. 
Published: November 2003

ABSTRACT

We
determined the frequency of DNA recombination between
Bombyx
mori
nucleopolyhedroviruses (BmNPVs) and
between BmNPV and the closely related
Autographa californica
NPV (Ac
M
NPV) in BmN cells, Sf-21 cells, and larvae of
Heliothis virescens
. The BmN cells were coinfected with two
BmNPVs, one with a mutation at the polyhedrin gene (
polh
)
locus and a second carrying a
lacZ
gene marker cassette.
Eleven different BmNPV mutants carrying the
lacZ
gene marker
at various distances (1.4 to 61.7 kb) from
polh
were used for
the coinfections. The Sf-21 cells and larvae of
H. virescens
were coinfected with wild-type Ac
M
NPV and 1 of the 11
lacZ
-marked BmNPV mutants. In BmN cells, high-frequency
recombination was detected as early as 15 h postcoinfection
but not at 12 h postcoinfection. At 18 h
postcoinfection, the mean frequency of recombination ranged between
20.0 and 35.4% when the
polh
and
lacZ
marker
genes were separated by at least 9.7 kb. When these marker genes were
separated by only 1.4 kb, the mean frequency of recombination was
2.7%. In BmN cells, the mean recombination frequency between two
BmNPVs increased only marginally when the multiplicity of infection of
each virus was increased 10-fold. In Sf-21 cells and the larvae of
H. virescens
, the recombination frequency between BmNPV and
Ac
M
NPV was ≤1.0%. Ac
M
NPV DNA
replication occurred normally after the coinfection of Sf-21 cells.
BmNPV DNA replication, however, was not detected, indicating that
normal DNA replication by both viruses is required for high-frequency
recombination.
Evolution of Human Calicivirus RNA In Vivo:Accumulation of Mutations in the Protruding P2 Domain of the CapsidLeads to Structural Changes and Possibly a NewPhenotype
Authors: Nilsson Mikael , Hedlund Kjell-Olof , Thorhagen Margareta , Larson Göran , Johansen Kari , Ekspong Anders , Svensson Lennart 
Published: November 2003

ABSTRACTIn the present study we report on evolution of calicivirus RNA from a patient with chronic diarrhea (i.e., lasting >2 years) and viral shedding. Partial sequencing of open reading frame 1 (ORF1) from 12 consecutive isolates revealed shedding of a genogroup II virus with relatively few nucleotide changes during a 1-year period. The entire capsid gene (ORF2) was also sequenced from the same isolates and found to contain 1,647 nucleotides encoding a protein of 548 amino acids with similarities to the Arg320 and Mx strains. Comparative sequence analysis of ORF2 revealed 32 amino acid changes during the year. It was notable that the vast majority of the cumulative amino acid changes (8 of 11) appeared within residues 279 to 405 located within the hypervariable domain (P2) of the capsid protein and hence were subject to immune pressure. An interesting and novel observation was that the accumulated amino acid changes in the P2 domain resulted in predicted structural changes, including disappearance of a helix structure, and thus a possible emergence of a new phenotype.FUT2gene polymorphism characterization revealed that the patient is heterozygous at nucleotide 428 and thus Secretor+, a finding in accordance with the hypothesis ofFUT2gene polymorphism and calicivirus susceptibility. To our knowledge, this is the first report of RNA evolution of calicivirus in a single individual, and our data suggest an immunity-driven mechanism for viral evolution. We also report on chronic virus excretion, immunoglobulin treatment, and modification of clinical symptoms; our observations from these studies, together with theFUT2gene characterization, may lead to a better understanding of calicivirus pathogenesis.
Different Heparan Sulfate Proteoglycans Serve asCellular Receptors for HumanPapillomaviruses
Authors: Shafti-Keramat Saeed , Handisurya Alessandra , Kriehuber Ernst , Meneguzzi Guerrino , Slupetzky Katharina , Kirnbauer Reinhard 
Published: November 2003

ABSTRACT
Papillomaviruses
replicate in stratified epithelia of skin and mucosa. Infection with
certain human papillomavirus (HPV) types is the main cause of
anogenital neoplasia, in particular cervical cancer. Early events of
papillomavirus infectivity are poorly understood. While heparan sulfate
proteoglycans (HSPGs) mediate initial binding to the cell surface, the
class of proteins carrying heparan sulfates has not been defined. Here
we examined two processes of papillomavirus infection, attachment of
virus-like particles (VLP) to cells and infection with authentic HPV
type 11 (HPV11) virions. Of the HSPGs, syndecan-1 is the major
epithelial form and is strongly upregulated in wound edge
keratinocytes. We employed K562 cells, which lack HSPGs except minor
amounts of endogenous betaglycan, and stable clones that express cDNAs
of syndecan-1, syndecan-4, or glypican-1. Binding of VLP correlated
with levels of heparan sulfate on the cell surface. Parental K562 bound
HPV16 VLP weakly, whereas all three K562 transfectants demonstrated
enhanced binding, with the highest binding capacity observed for
syndecan-1-transfected cells, which also expressed the most HSPG. For
HPV11 infectivity assays, a high virion inoculum was required to infect
K562 cells, whereas ectopic expression of syndecan-1 increased
permissiveness eightfold and expression of syndecan-4 or glypican-1
fourfold. Infection of keratinocytes was eliminated by treatment with
heparitinase, but not phospholipase C, further implicating the syndecan
family of integral membrane proteins as receptor proteins. Human
keratinocytes with a homozygous deletion of α6 integrin are
permissive for HPV11 infection. These results indicate that several
HSPGs can serve as HPV receptors and support a putative role for
syndecan-1, rather than α6 integrin, as a primary receptor
protein in natural HPV infection of
keratinocytes.
HerpesSimplex Virus 1 Mutant in Which the ICP0 HUL-1 E3 Ubiquitin Ligase SiteIs Disrupted Stabilizes cdc34 but Degrades D-Type Cyclins andExhibits DiminishedNeurotoxicity
Authors: Hagglund Ryan , Roizman Bernard 
Published: November 2003

ABSTRACT
Herpes
simplex virus type 1 (HSV-1) infected cell protein 0 (ICP0) is a
multifunctional protein that functions as a promiscuous transactivator
and promotes the degradation of multiple cellular proteins. In vitro
studies indicated that it encodes two physically separated functional
E3 ubiquitin ligase domains. One, designated herpesvirus ubiquitin
ligase 1 (HUL-1), maps to a region encoded by exon 3 and is contained
between residues 543 and 680. Deletion of amino acids 621 to 625
abolishes this activity. The second, designated HUL-2, maps to the RING
finger domain present in ICP0 encoded by exon 2. Earlier studies have
shown that ICP0 stabilizes cyclins D1 and D3, and several lines of
investigation led to the hypothesis that this function of ICP0 is the
consequence of degradation of the E2 enzyme cdc34, known to be involved
in the proteasome-dependent degradation of D-type cyclins. Consistent
with this hypothesis, we have previously shown that cdc34 physically
interacts with ICP0 at or near aspartate 199 and at amino acids 621 to
625 and that the former site is required for effective ubiquitylation
and degradation of cdc34. Furthermore, the ICP0 HUL-1 domain promotes
the polyubiquitination of cdc34 in vitro. If the mechanism by which
D-type cyclins are salvaged in wild-type-infected cells is dependent on
polyubiquitination and consequent destruction of cdc34, than the mutant
virus R6701, which was constructed for these studies and lacks ICP0
residues 621 to 625, should destabilize the D cyclins and preclude the
degradation of cdc34. We report that ICP0 residues 621 to 625 are
essential for degradation of cdc34 in infected cells and for the
ICP0-mediated stabilization of D-type cyclins, that a mutation that
specifically disrupted the ring finger domain of the HUL-2 site had no
effect on the degradation of cdc34 in infected cells, and that deletion
of ICP0 residues 621 to 625 decreased the replicative capacity of the
virus in growth-arrested but not in dividing cells and resulted in
diminished pathogenicity on intracerebral inoculation of mice. We
conclude that the ICP0 HUL-1 domain acts in infected cells to degrade
cdc34 and that this function requires the interaction of cdc34 with
sequences in exons 2 and 3 but does not involve the HUL-2 RING finger
E3
domain.
Mechanisms Governing Maintenance of Cdk1/Cyclin B1Kinase Activity in Cells Infected with HumanCytomegalovirus
Authors: Sanchez Veronica , McElroy Anita K. , Spector Deborah H. 
Published: November 2003

ABSTRACT
Previous
work has demonstrated dysregulation of key cell cycle components in
human cytomegalovirus (HCMV)-infected human fibroblasts, resulting in
cell cycle arrest (F. M. Jault, J.-M. Jault, F. Ruchti,
E. A. Fortunato, C. L. Clark, J. Corbeil,
D. D. Richman, and D. H. Spector, J.
Virol. 69:6697-6704, 1995). The activation of the mitotic
kinase Cdk1/cyclin B, which was detected as early as 8 h
postinfection (p.i.) and maintained throughout the time course, was
particularly interesting. To understand the mechanisms underlying the
induction of this kinase activity, we have examined the pathways that
regulate the activation of Cdk1/cyclin B1 complexes. The accumulation
of the cyclin B1 subunit in HCMV-infected cells is the result of
increased synthesis and reduced degradation of the protein. In
addition, the catalytic subunit, Cdk1, accumulates in its active form
in virus-infected cells. The decreased level of the
Tyr15-phosphorylated form of Cdk1 in virus-infected fibroblasts is due
in part to the down-regulation of the expression and activity of the
Cdk1 inhibitory kinases Myt1 and Wee1. Increased degradation of Wee1
via the proteasome also accounts for its absence at 24 h p.i.
At late times, we observed accumulation of the Cdc25 phosphatases that
remove the inhibitory phosphates from Cdk1. Interestingly, biochemical
fractionation studies revealed that the active form of Cdk1, a fraction
of total cyclin B1, and the Cdc25 phosphatases reside predominantly in
the cytoplasm of infected cells. Collectively, these data suggest that
the maintenance of Cdk1/cyclin B1 activity observed in HCMV-infected
cells can be explained by three mechanisms: the accumulation of cyclin
B1, the inactivation of negative regulatory pathways for Cdk1, and the
accumulation of positive factors that promote Cdk1
activity.