bluuebunny/crossref_metadata_2025 huggingface dataset:
Disk Diffusion-Based Methods for Determining
Candida parapsilosis
Susceptibility to Anidulafungin
Authors: Odabasi Zekaver , Paetznick Victor , Goldstein Beth P. , Rex John H. , Ostrosky-Zeichner Luis
Published: August 2003

Zone diameters for anidulafungin by disk diffusion for 139 isolates of
C. parapsilosis
were compared with MICs by NCCLS M27-A2 broth microdilution. The comparison was poor unless the disks were prepared by dissolving anidulafungin in 1% dimethyl sulfoxide plus 0.1% Tween 80 and testing on Mueller-Hinton agar flooded with glucose and methylene blue.
Real-Time PCR as a Versatile Tool for Investigating the Susceptibility of Human Herpesvirus 6 to Antiviral Agents
Authors: Macé Muriel , Manichanh Chaysavanh , Bonnafous Pascale , Précigout Stéphanie , Boutolleau David , Gautheret-Dejean Agnès , Agut Henri
Published: August 2003

A quantitative real-time PCR assay was developed for the determination of antiviral drug susceptibility and growth kinetics of human herpesvirus 6. The susceptibility and fitness of a sensitive strain, HST, and its ganciclovir-resistant derivative, GCVR1, were then characterized, leading us to conclude that the mutations of this latter virus did not alter its fitness significantly.
In Vitro Activities of a New Lipopeptide, HMR 1043, against Susceptible and Resistant Gram-Positive Isolates
Authors: Bemer Pascale , Juvin Marie-Emmanuelle , Bryskier Andre , Drugeon Henri
Published: August 2003

The purpose of this study was to compare the activity of HMR 1043 with those of daptomycin and teicoplanin against gram-positive isolates. Susceptibility tests were performed for 52 strains, 26 parental strains, including staphylococcal, streptococcal, enterococcal, and listerial strains, and 26 HMR 1043-resistant mutants obtained from parental strains by using the Szybalski method. Agar dilution and disk diffusion susceptibility tests were performed by the procedures outlined by the NCCLS. HMR 1043 demonstrated good activity against susceptible and resistant gram-positive bacteria. The activity of HMR 1043 in vitro was less influenced by the presence of calcium ions than that of daptomycin. Susceptibility test breakpoints were not defined because of the poor correlation coefficients obtained with the different disks tested.
Roles of TolC-Dependent Multidrug Transporters of
Escherichia coli
in Resistance to β-Lactams
Authors: Nishino Kunihiko , Yamada Junko , Hirakawa Hidetada , Hirata Takahiro , Yamaguchi Akihito
Published: August 2003

AcrAB exports some β-lactam antibiotics in the periplasm out of cells via an outer-membrane channel, TolC. It has been reported that eight drug transporters in
Escherichia coli
cooperate with TolC. In this study, the roles of the drug exporters of
E. coli
in β-lactam resistance were examined. We found that five of five resistance-nodulation-cell division-type drug exporters confer β-lactam antibiotic resistance, while no other drug exporters confer any β-lactam resistance even when they cooperate with TolC.
MtuA, a Lipoprotein Receptor Antigen from
Streptococcus uberis
, Is Responsible for Acquisition of Manganese during Growth in Milk and Is Essential for Infection of the Lactating Bovine Mammary Gland
Authors: Smith Amanda J. , Ward Philip N. , Field Terence R. , Jones Catherine L. , Lincoln Ruth A. , Leigh James A.
Published: August 2003

A mutant strain of
Streptococcus uberis
(AJS001) that was unable to grow in bovine milk was isolated following random insertional mutagenesis. The level of growth in milk was restored to that of the parental strain (strain 0140J) following addition of MnSO
4
but not following addition of other metal ions. The mutant contained a single insertion within
mtuA
, a homologue of
mtsA
and
psaA
, which encode metal-binding proteins in
Streptococcus pyogenes
and
Streptococcus pneumoniae
, respectively. Strain AJS001 was unable to infect any of eight quarters on four dairy cows following intramammary challenge with 10
5
CFU. Bacteria were never recovered directly from milk of these animals but were detected following enrichment in Todd-Hewitt broth in three of eight milk samples obtained within 24 h of challenge. The animals showed no inflammatory response and no signs of mastitis. Three mammary quarters on two different animals simultaneously challenged with 600 CFU of the parental strain, strain 0140J, became colonized, shed high numbers of
S. uberis
organisms in milk, displayed a marked inflammatory response to infection, and showed overt signs of mastitis. These data indicate that
mtuA
was required for efficient uptake of Mn
2+
during growth in bovine milk and infection of the lactating bovine mammary gland.
Role of Toll-Like Receptor 4 in Macrophage Activation and Tolerance duringSalmonella entericaSerovar Typhimurium Infection
Authors: Li Qian , Cherayil Bobby J.
Published: August 2003

Toll-like receptors (TLRs) play an important role in the innate immune response, particularly in the initial interaction between the infecting microorganism and phagocytic cells, such as macrophages. We investigated the role of TLR4 during infection of primary murine peritoneal macrophages withSalmonella entericaserovar Typhimurium. We found that macrophages from the C3H/HeJ mouse strain, which carries a functionally inactive Tlr4 gene, exhibit marked impairment of tumor necrosis factor alpha (TNF-α) secretion in response toS. entericaserovar Typhimurium infection. However, activation of extracellular growth factor-regulated kinase and NF-κB signaling pathways was relatively unaffected, as was increased expression of TNF-α mRNA. Furthermore, macrophage tolerance, which is associated with increased expression of the NF-κB p50 and p52 subunits, was induced byS. entericaserovar Typhimurium even in the absence of functional TLR4. These results indicate that during infection of macrophages byS. entericaserovar Typhimurium, TLR4 signals are required at a posttranscriptional step to maximize secretion of TNF-α. Signals delivered by pattern recognition receptors other than TLR4 are sufficient for the increased expression of the TNF-α transcript and at least some genes associated with macrophage tolerance.
Interactions of
Campylobacter jejuni
Cytolethal Distending Toxin Subunits CdtA and CdtC with HeLa Cells
Authors: Lee Robert B. , Hassane Duane C. , Cottle Daniel L. , Pickett Carol L.
Published: August 2003

Campylobacter jejuni
produces a toxin, called cytolethal distending toxin (CDT), which causes direct DNA damage leading to invocation of DNA damage checkpoint pathways. The affected cells arrest in G
1
or G
2
and eventually die. CDT consists of three protein subunits, CdtA, CdtB, and CdtC, with CdtB recently identified as a nuclease. However, little is known about the functions of CdtA or CdtC. In this work, enzyme-linked immunosorbent assay-based experiments were used to show, for the first time, that both CdtA and CdtC bound with specificity to the surface of HeLa cells, whereas CdtB did not. Varying the order of the addition of subunits for reconstitution of the holotoxin had no effect on activity. In addition, mutants containing deletions of conserved regions of CdtA and CdtC were able to bind to the surface of HeLa cells but were not able to participate in holotoxin assembly. Finally, both Cdt mutant subunits were able to effectively compete with CDT holotoxin in the HeLa cell binding assay.
Increased Mortality and Dysregulated Cytokine Production in Tumor Necrosis Factor Receptor 1-Deficient Mice following SystemicKlebsiella pneumoniaeInfection
Authors: Moore Thomas A. , Perry Michelle L. , Getsoian Andrew G. , Monteleon Christine L. , Cogen Anna L. , Standiford Theodore J.
Published: August 2003

A significant clinical complication of pulmonary infections withKlebsiella pneumoniaeis peripheral blood dissemination, resulting in a systemic infection concurrent with the localized pulmonary infection. In this context, little is known about the role of tumor necrosis factor receptor 1 (TNFR1)-mediated innate immune responses during systemicKlebsiellainfections. Mice lacking TNFR1 were significantly more susceptible toKlebsiella-induced mortality following intravenous inoculation. Bacterial clearance was impaired in TNFR1-deficient mice at early times following infection. Unexpectedly, bacterial burdens at the onset of mortality (days 2 to 3 postinfection) were not higher in mice lacking TNFR1. However, elevated production of liver-associated proinflammatory cytokines (interleukin-12, tumor necrosis factor alpha [TNF-α[, and gamma interferon [IFN-γ]) and chemokines (MIP-1α, MIP-2, and MCP-1) was observed within the first 24 h of infection. Additionally, excessive plasma-associated IFN-γ was also observed late in the course of infection (day 3). Spleen cells from day-3 infected TNFR1-deficient mice secreted markedly enhanced levels of IFN-γ when cultured in vitro. Additionally, there was a marked increase in the total number of activated lymphocyte subsets as indicated by CD69 upregulation. A notable exception was the sharp decrease in the frequency of splenic NK T cells in infected TNFR1 knockout (KO) mice. Anti-TNF-α therapy in TNFR1 KO mice significantly reduced chemokine production and liver injury. Combined, these data indicate a dysregulated antibacterial host response following intravenousKlebsiellainfection in the absence of TNFR1 signaling, resulting in heightened cytokine production and hyperactivation of specific splenic lymphocyte subsets.
A DNA Prime-
Mycobacterium bovis
BCG Boost Vaccination Strategy for Cattle Induces Protection against Bovine Tuberculosis
Authors: Skinner Margot A. , Buddle Bryce M. , Wedlock D. Neil , Keen Denise , de Lisle Geoffrey W. , Tascon Ricardo E. , Candido Ferraz Jose , Lowrie Douglas B. , Cockle Paul J. , Vordermeier H. Martin , Glyn Hewinson R.
Published: August 2003

The variable efficacy of bacillus Calmette-Guérin (
Mycobacterium bovis
BCG) in protecting humans and cattle against tuberculosis has prompted a search for a more effective vaccination regimen. A prime-boost strategy was investigated in cattle naturally sensitized to environmental mycobacteria by using a combination of three DNA vaccines coding for Hsp 65, Hsp 70, and Apa for priming, followed by a boost with BCG prior to experimental challenge with virulent
M. bovis.
Controls were vaccinated with DNA or BCG alone or were not vaccinated. The immune responses were monitored throughout the study, and protection was assessed based on reductions in the numbers of lesions and viable mycobacteria in lymph node samples. Vaccination with BCG alone or with a DNA prime-BCG boost regimen induced high levels of antigen-specific gamma interferon (IFN-γ) in whole-blood cultures. In the prime-boost group there were fewer animals with severe lung lesions, fewer lymph nodes with lesions per animal, a smaller proportion of animals with lesions, lower mean lung and lymph node lesion scores, and less
M. bovis
isolated from retropharyngeal and thoracic lymph nodes compared to the results obtained for the nonvaccinated animals. The prime-boost regimen induced significant enhancement of protection in six parameters, compared with significant enhancement of protection in only two parameters for BCG alone. In addition, following challenge, in vitro IFN-γ responses against ESAT-6 and CFP-10, as well as bovine tuberculin-induced skin test and in vitro IFN-γ responses, were identified as immunological markers that predicted protection. The use of the prime-boost strategy suggested that a combination of vaccines may be better than a single vaccine for protection against tuberculosis.
Effect of Transcription Factor GATA-2 on Phagocytic Activity of Alveolar Macrophages fromPneumocystis carinii-Infected Hosts
Authors: Lasbury Mark E. , Tang Xing , Durant Pamela J. , Lee Chao-Hung
Published: August 2003

Alveolar macrophages fromPneumocystis carinii-infected hosts are defective in phagocytosis (W. Chen, J. W. Mills, and A. G. Harmsen, Int. J. Exp. Pathol. 73:709-720, 1992; H. Koziel et al., J. Clin. Investig. 102:1332-1344, 1998). Experiments were performed to determine whether this defect is specific forP. cariniiorganisms. The results showed that these macrophages were unable to phagocytose bothP. cariniiorganisms and fluorescein isothiocyanate (FITC)-conjugated latex beads, indicating that alveolar macrophages fromP. carinii-infected hosts have a general defect in phagocytosis. To determine whether this defect correlates with the recently discovered down-regulation of the GATA-2 transcription factor gene duringP. cariniiinfection, alveolar macrophages from dexamethasone-suppressed or healthy rats were treated with anti-GATA-2 oligonucleotides and then assayed for phagocytosis. Aliquots of the alveolar macrophages were also treated with the sense oligonucleotides as the control. Cells treated with the antisense oligonucleotides were found to have a 46% reduction in phagocytosis ofP. cariniiorganisms and a 65% reduction in phagocytosis of FITC-latex beads compared to those treated with the sense oligonucleotides. To determine whether the defect in phagocytosis in alveolar macrophages fromP. carinii-infected hosts can be corrected by overexpression of GATA-2, a plasmid containing the rat GATA-2 gene in the sense orientation driven by the cytomegalovirus (CMV) promoter was introduced into alveolar macrophages fromP. carinii-infected rats. Aliquots of the same cells transfected with a plasmid containing GATA-2 in the antisense orientation relative to the CMV promoter served as the control. Alveolar macrophages treated with the sense GATA-2 expression construct were found to increase their phagocytic activity by 66% in phagocytosis ofP. cariniiorganisms and by 280% in phagocytosis of FITC-latex beads compared to those that received the antisense GATA-2 construct. The results of this study indicate that GATA-2 plays an important role in the regulation of phagocytosis in alveolar macrophages duringP. cariniiinfection.
Importance of a Developmentally Regulated Pheromone Receptor of
Cryptococcus neoformans
for Virulence
Authors: Chang Yun C. , Miller Georgina F. , Kwon-Chung K. J.
Published: August 2003

Cryptococcus neoformans
is the etiologic agent of cryptococcosis. Two mating types exist in this fungus,
MAT
α and
MAT
a
. The
CPR
a
gene of
C. neoformans
is a
MAT
a
strain-specific gene and encodes a putative seven-transmembrane domain pheromone receptor. Unlike the other reported fungal pheromone receptors,
CPR
a
shows functional diversity. Deletion of
CPR
a
drastically affects mating efficiency but does not abolish mating.
CPR
a
expression is developmentally regulated and is not affected by deletion of the transcriptional regulator
STE12
a
. The expression of
CPR
a
is markedly increased by shifting cultures from liquid to solid media.
CPR
a
also plays a significant role in virulence. Δ
cpr
a
cells produce smaller capsules in the brains of mice than the wild-type cells, and the mice infected with Δ
cpr
a
survive significantly longer than those receiving the wild-type strain. Our results suggest that the
MAT
a
pheromone receptor of
C. neoformans
is not only required for mating but also important for survival and growth of the fungus in host tissue.
Effect of Codon Optimization on Expression Levels of a Functionally Folded Malaria Vaccine Candidate in Prokaryotic and Eukaryotic Expression Systems
Authors: Yadava Anjali , Ockenhouse Christian F.
Published: August 2003

We have produced two synthetic genes that code for the F2 domain located within region II of the 175-kDa
Plasmodium falciparum
erythrocyte binding antigen (EBA-175) to determine the effects of codon alteration on protein expression in homologous and heterologous host systems. EBA-175 plays a key role in the process of merozoite invasion into erythrocytes through a specific receptor-ligand interaction. The F2 domain of EBA-175 is the ligand that binds to the glycophorin A receptor on human erythrocytes and is therefore a target of vaccine development efforts. We designed synthetic genes based on
P. falciparum
,
Escherichia coli
, and
Pichia
codon usage and expressed recombinant F2 in
E. coli
and
Pichia pastoris.
Compared to the expression of the native F2 sequence, conversion to prokaryote (
E. coli
)- or eukaryote (
Pichia
)-based codon usage dramatically improved the levels of recombinant protein expression in both
E. coli
and
P. pastoris
. The majority of the protein expressed in
E. coli
, however, was produced as inclusion bodies. The protein expressed in
P. pastoris
, on the other hand, was expressed as a secreted, soluble protein. The
P. pastoris-
produced protein was superior to that produced in
E. coli
based on its ability to bind to red blood cells. Consistent with these observations, the antibodies generated against the
Pichia
-produced protein prevented the binding of recombinant EBA to red blood cells. These antibodies recognize EBA-175 present on merozoites as well as in sporozoites by immunofluorescence. Our results suggest that the
Pichia
-based EBA-F2 vaccine construct has further potential to be developed for clinical use.
Stimulation of T-Helper Cell Gamma Interferon and Immunoglobulin G Responses Specific forBabesia bovisRhoptry-Associated Protein 1 (RAP-1) or a RAP-1 Protein Lacking the Carboxy-Terminal Repeat Region Is Insufficient To Provide Protective Immunity against VirulentB. bovisChallenge
Authors: Norimine Junzo , Mosqueda Juan , Suarez Carlos , Palmer Guy H. , McElwain Terry F. , Mbassa Gabriel , Brown Wendy C.
Published: August 2003

Rhoptry-associated protein 1 (RAP-1) is a targeted vaccine antigen forBabesia bovisandBabesia bigeminainfections of cattle. The 60-kDaB. bovisRAP-1 is recognized by antibodies and T lymphocytes from cattle that recovered from infection and were immune to subsequent challenge. Immunization with native or recombinant protein was reported to reduce parasitemias in challenged animals. We recently reported that the NT domain ofB. bovisRAP-1 contained immunodominant T-cell epitopes, whereas the repeat-rich CT domain was less immunostimulatory for T lymphocytes from cattle immune toB. bovis.The present study was therefore designed to test the hypothesis that the NT region of RAP-1, used as a vaccine with interleukin-12 and RIBI (catalog no. R-730; RIBI Immunochem Research, Inc., Hamilton, Mont. [now Corixa, Seattle, Wash.]) adjuvant to induce a type 1 response, would prime calves for antibody and T-helper cell responses comparable to or greater than those induced by full-length RAP-1 containing the C-terminal repeats. Furthermore, a type 1 immune response to RAP-1 was hypothesized to induce protection against challenge. Following four inoculations of either recombinant full-length RAP-1 or RAP-1 NT protein, RAP-1-specific immunoglobulin G (IgG) titers, T-lymphocyte proliferation, and gamma interferon production were similar. Similar numbers of NT region peptides were recognized. However, in spite of the presence of strong RAP-1-specific IgG and CD4+-T-lymphocyte responses that were recalled upon challenge, neither antigen stimulated a protective immune response. We conclude that successful priming of calves with recombinant RAP-1 and adjuvants that elicit strong Th1 cell and IgG responses is insufficient to protect calves against virulentB. bovischallenge.
Borrelia burgdorferi
Population Dynamics and Prototype Gene Expression during Infection of Immunocompetent and Immunodeficient Mice
Authors: Hodzic Emir , Feng Sunlian , Freet Kim J. , Barthold Stephen W.
Published: August 2003

The population dynamics of
Borrelia burgdorferi
were quantified by real-time PCR targeting the
flaB
gene in skin (inoculation site, noninoculation site, and ear), heart (heart base and ventricle), quadriceps muscle, and the tibiotarsal joint at 1, 2, 4, 6, and 8 weeks after intradermal inoculation in C3H and C3H-
scid
mice. In addition, RNA transcription was assessed for several prototype genes, including
flaB
,
ospA
,
ospC
,
dbpA
,
arp
,
vlsE
,
fbp
,
oppA-2
, and
p37-42
. Spirochete numbers were equivalent in C3H and C3H-
scid
mice at 1 or 2 weeks and then declined in C3H mice, but they continued to rise and then plateaued in C3H-
scid
mice. Gene transcription was likewise higher in C3H-
scid
mice than in C3H mice, particularly at 4 or more weeks of infection. Gene transcription showed variation among tissues, with the highest levels of transcription in heart and joint tissue, which are sites of inflammation.
A Novel Pathogenicity Island Integrated Adjacent to the
thrW
tRNA Gene of Avian Pathogenic
Escherichia coli
Encodes a Vacuolating Autotransporter Toxin
Authors: Parreira V. R. , Gyles C. L.
Published: August 2003

We report the complete nucleotide sequence and genetic organization of the Vat-encoding pathogenicity island (PAI) of avian pathogenic
Escherichia coli
strain Ec222. The 22,139-bp PAI is situated adjacent to the 3′ terminus of the
thrW
tRNA gene, has a G+C content of 41.2%, and includes a bacteriophage SfII integrase gene, mobile genetic elements, two open reading frames with products exhibiting sequence similarity to known proteins, and several other open reading frames of unknown function. The PAI encodes an autotransporter protein, Vat (vacuolating autotransporter toxin), which induces the formation of intracellular vacuoles resulting in cytotoxic effects similar to those caused by the VacA toxin from
Helicobacter pylori
. The predicted 148.3-kDa protein product possesses the three domains that are typical of serine protease autotransporters of
Enterobacteriaceae
: an N-terminal signal sequence of 55 amino acids, a 111.8-kDa passenger domain containing a modified serine protease site (ATSGSG), and a C-terminal outer membrane translocator of 30.5 kDa. Vat has 75% protein homology with the hemagglutinin Tsh, an autotransporter of avian pathogenic
E. coli
. A
vat
deletion mutant of Ec222 showed no virulence in respiratory and cellulitis infection models of disease in broiler chickens. We conclude that the newly described PAI and Vat may be involved in the pathogenicity of avian septicemic
E. coli
strain Ec222 and other avian pathogenic
E. coli
strains.
Preparation and Characterization of Group A Meningococcal Capsular Polysaccharide Conjugates and Evaluation of Their Immunogenicity in Mice
Authors: Jin Zhigang , Chu Chiayung , Robbins John B. , Schneerson Rachel
Published: August 2003

Epidemic and endemic meningitis caused by group ANeisseria meningitidisremains a problem in sub-Saharan Africa. Although group A meningococcal capsular polysaccharide (GAMP) vaccine confers immunity at all ages, the improved immunogenicity of a conjugate and its compatibility with the World Health Organization's Extended Program on Immunization offers advantages over GAMP alone. Conjugates of GAMP bound to bovine serum albumin (BSA) were synthesized, characterized, and evaluated for their immunogenicities in mice. Two methods, involving adipic acid dihydrazide (ADH) as a linker, were used. First, ADH was bound to GAMP activated with cyanogen bromide (CNBr) or with 1-cyano-4(dimethylamino)-pyridinium tetrafluoroborate (CDAP) to form GAMPCNBrAHand GAMPCDAPAH. These derivatives were bound to BSA by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to form GAMPCNBrAH-BSA and GAMPCDAPAH-BSA. Second, ADH was bound to BSA with EDC to formAHBSA.AHBSA was bound to activated GAMP to form GAMPCNBr-AHBSA and GAMPCDAP-AHBSA. The yield of GAMPCDAP-AHBSA (35 to 40%) was higher than those of the other conjugates (5 to 20%). GAMP conjugates elicited immunoglobulin G (IgG) anti-GAMP in all mice after three injections of 2.5 or 5.0 μg of GAMP: the geometric mean (GM) was highest in recipients of GAMPCDAP-AHBSA (11.40 enzyme-linked immunosorbent assay units). Although the difference was not statistically significant, the 5.0-μg dose elicited a higher GM IgG anti-GAMP than the 2.5-μg dose. Low levels of anti-GAMP were elicited by GAMP alone. GAMPCDAP-AHBSA elicited bactericidal activity roughly proportional to the level of IgG anti-GAMP.
SarT Influences
sarS
Expression in
Staphylococcus aureus
Authors: Schmidt Katherine A. , Manna Adhar C. , Cheung Ambrose L.
Published: August 2003

Staphylococcus aureus
is a gram-positive pathogen that is capable of expressing a variety of virulence proteins in response to environmental signals. Virulence protein expression in
S. aureus
is controlled by a network of regulatory loci including
sarA
and
agr
. The
sarA/agr
network is associated with the expression of cell wall-associated adhesins during exponential growth and the expression of secreted enzymes and toxins in the transition to post-exponential growth. A number of
sarA
homologs, including
sarT
and
sarS
, have been identified in the
S. aureus
genome. Previous studies have shown that
sarA
influences expression of both
sarT
and
sarS
in the global regulatory network. SarS has been shown to bind to the
spa
promoter to induce expression of protein A. SarT, one of the SarA homologs that represses
hla
expression and is repressible by SarA and
agr
, was found to induce
sarS
expression in this report. Northern blot analysis of
sarS
and
spa
expression in
S. aureus
RN6390, and the isogenic
sarT
,
sarT sarA
, and
sarT agr
mutants showed that while
sarA
regulated
spa
expression directly, the
agr
locus used
sarT
as an intermediary to regulate
sarS
, thus leading to
spa
repression in
agr
-activated cells. Gel shift and footprinting analysis showed that SarT binds to the
sarS
promoter, indicating that the interaction of the
sarT
gene product with the upstream region of
sarS
is likely direct. Induction of
sarS
and
spa
by SarT in

agr
+

strains was confirmed by a tetracycline-inducible system to titrate
sarT
expression.
Porphyromonas gingivalisInfection in Pregnant Mice Is Associated with Placental Dissemination, an Increase in the Placental Th1/Th2 Cytokine Ratio, and Fetal Growth Restriction
Authors: Lin Dongming , Smith Mary Alice , Elter John , Champagne Catherine , Downey Christine Lynn , Beck James , Offenbacher Steven
Published: August 2003

Our previous animal studies showed that maternalPorphyromonas gingivalisinfection in a subcutaneous chamber is associated with hepatic and uterine translocation, as well as systemic induction of maternal inflammatory responses, both of which were associated with fetal growth restriction (FGR). However,P. gingivalis-challenged dams had fetuses with either FGR (2 standard deviations below mean weight of nonchallenged dams) or normal weight. Therefore, the objective of this study was to determine whether maternal infection withP. gingivaliscompromises normal fetal development via direct placental invasion and induction of fetus-specific placental immune responses characterized by a proinflammatory Th1-type cytokine profile.P. gingivalis-specific DNA was detected in placentas and fetuses of FGR and normal littermates fromP. gingivalis-infected dams. Th1- and Th2-type cytokine mRNA as well as tumor necrosis factor alpha and transforming growth factor β2 mRNA were examined in placental tissue by using reverse transcription-PCR to determine Th1/Th2 ratios. For eight litters containing both normal-weight and FGR fetuses,P. gingivalisDNA was detected only in the placentas of FGR fetuses. All fetuses and all amniotic fluid samples from infected and control dams were negative forP. gingivalisDNA. mRNA levels of gamma interferon and interleukin-2 (IL-2) were significantly increased in placentas of FGR fetuses, while expression of IL-10 was significantly decreased in the same group. These data indicate that, inP. gingivalis-challenged dams, within each litter there is placenta-specific translocation ofP. gingivalisthat results in growth restriction of the targeted fetus, which is associated with a shift in the placental Th1/Th2 cytokine balance.
Vaccination with Novel Immunostimulatory Adjuvants against Blood-Stage Malaria in Mice
Authors: Su Zhong , Tam Mi-Fong , Jankovic Dragana , Stevenson Mary M.
Published: August 2003

An important aspect of malaria vaccine development is the identification of an appropriate adjuvant which is both capable of stimulating a protective immune response and safe for use by humans. Here, we investigated the feasibility of using novel immunostimulatory molecules as adjuvants combined with a crude antigen preparation and coadsorbed to aluminum hydroxide (alum) as a vaccine against blood-stagePlasmodium chabaudiAS malaria. Prior to challenge infection, immunization of genetically susceptible A/J mice with the combination of malaria antigen plus recombinant interleukin-12 (IL-12) in alum induced a Th1 immune response with production of high levels of gamma interferon (IFN-γ) and diminished IL-4 levels by spleen cells stimulated in vitro with parasite antigen compared to mice immunized with antigen alone, antigen in alum, or antigen plus IL-12. Mice immunized with malaria antigen plus recombinant IL-12 in alum had high levels of total malaria-specific antibody and immunoglobulin G2a. Compared to unimmunized mice, immunization with antigen plus IL-12 in alum induced the highest level of protective immunity against challenge infection withP. chabaudiAS, which was evident as a significantly decreased peak parasitemia level and 100% survival. Protective immunity was dependent on CD4+T cells, IFN-γ, and B cells and was long-lasting. Replacement of IL-12 as an adjuvant by synthetic oligodeoxynucleotides (ODN) containing CpG motifs induced a similar level of vaccine-induced protection against challenge infection withP. chabaudiAS. These results illustrate that it is possible to enhance the potency of a crude malaria antigen preparation delivered in alum by inclusion of immunostimulatory molecules, such as IL-12 or CpG-ODN.
Cytotoxic Necrotizing Factor 1 ofEscherichia coliStimulates Rho/Rho-Kinase-Dependent Myosin Light-Chain Phosphorylation without Inactivating Myosin Light-Chain Phosphatase in Endothelial Cells
Authors: Essler Markus , Linder Stefan , Schell Barbara , Hüfner Katharina , Wiedemann Agnès , Randhahn Katharina , Staddon James M. , Aepfelbacher Martin
Published: August 2003

Cytotoxic necrotizing factor 1 (CNF-1) is an exotoxin ofEscherichia colithat constitutively activates the GTPases Rho, Rac, and CDC42. Stimulation of Rho was shown to enhance myosin light-chain (MLC) phosphorylation via Rho kinase-mediated inhibition of MLC phosphatase in endothelial cells. Here we report that 3 h after CNF stimulation of endothelial cells, RhoA was activated and MLC phosphorylation was increased in a Rho/Rho-kinase-dependent manner, but no decrease in MLC phosphatase activity could be detected. Despite continuous RhoA activation, MLC phosphatase activity was doubled after 24 h of CNF stimulation, and this coincided with decreased MLC phosphorylation and cell spreading. Rac was also activated at 3 to 24 h but did not contribute to MLC phosphorylation, and its amount gradually decreased in the CNF-stimulated cells. CDC42Hs was not activated above control values by CNF. These results suggest that CNF can induce specific decoupling (Rho kinase from MLC phosphatase) and deactivation events in Rho GTPase signaling, potentially reflecting cellular protection mechanisms against permanently active Rho GTPases.
Influence of Pregnancy on the Pathogenesis of Listeriosis in Mice Inoculated Intragastrically
Authors: Hamrick Terri S. , Horton John R. , Spears Patricia A. , Havell Edward A. , Smoak Ida W. , Orndorff Paul E.
Published: August 2003

Pregnancy increases the risk of listeriosis, a systemic disease caused byListeria monocytogenes. However, there is incomplete agreement on the reasons for this increased risk. We examined two features of listeriosis in gravid and nongravid female mice following intragastric (gavage) inoculation, namely, (i) disease severity (measured by lethality) and (ii) listerial infectivity (measured by liver and spleen colonization levels up to 120 h postinoculation). Two listerial strains of differing serotype (1/2a and 4nonb) were initially employed. Neither strain produced a lethal infection in nonpregnant female mice (dose range, 106to 109CFU/mouse), and only the 4nonb strain produced lethalities in pregnant mice (dose range, 106to 108CFU/mouse). The 4nonb strain also produced a higher level of liver and spleen colonization than the 1/2a strain following gavage administration. (The two strains showed similar levels of colonization if parenterally administered.) Both strains were equally capable of binding to and forming plaques upon cultured mouse enterocytes. The ability of the 4nonb strain to produce a lethal infection in pregnant animals did not correlate with an increased incidence or level of liver and spleen colonization over that in nonpregnant females. However, the lethality rate did correlate well with the rate at which embryos and their surrounding decidual covering became infected, suggesting that intrauterine infection could be responsible for the increased disease severity in the gravid females.
GastricHelicobacterInfection Inhibits Development of Oral Tolerance to Food Antigens in Mice
Authors: Matysiak-Budnik Tamara , van Niel Guillaume , Mégraud Francis , Mayo Kathryn , Bevilacqua Claudia , Gaboriau-Routhiau Valérie , Moreau Marie-Christiane , Heyman Martine
Published: August 2003

The increase in the transcellular passage of intact antigens across the digestive epithelium infected withHelicobacter pylorimay interfere with the regulation of mucosal immune responses. The aim of this work was to study the capacity ofHelicobacterinfection to inhibit the development of oral tolerance or to promote allergic sensitization and the capacity of a gastro-protective agent, rebamipide, to interfere with these processes in mice. Oral tolerance to ovalbumin (OVA) was studied in 48 C3H/He 4-week-old mice divided into four groups: (i) OVA-sensitized mice; (ii) OVA-“tolerized” mice (that is, mice that were rendered immunologically tolerant); (iii)H. felis-infected, OVA-tolerized mice; (iv) andH. felis-infected, OVA-tolerized, rebamipide-treated mice. Oral sensitization to hen egg lysozyme (HEL) was studied in 48 mice divided into four groups: (i) controls; (ii) HEL-sensitized mice; (iii)H. felis-infected, HEL-sensitized mice; and (iv)H. felis-infected, HEL-sensitized, rebamipide-treated mice. Specific anti-OVA or anti-HEL immunoglobulin E (IgE) and IgG1/IgG2a serum titers were measured by enzyme-linked immunosorbent assay. Additionally, the capacity of rebamipide to interfere with antigen presentation and T-cell activation in vitro, as well as absorption of rebamipide across the epithelial monolayer, was tested.H. felisinfection led to the inhibition of oral tolerance to OVA, but rebamipide prevented this inhibitive effect ofH. felis. H. felisinfection did not enhance the sensitization to HEL, but rebamipide inhibited the development of this sensitization. Moreover, rebamipide inhibited in a dose-dependent manner antigen presentation and T-cell activation in vitro and was shown to be able to cross the epithelium at a concentration capable of inducing this inhibitory effect. We conclude thatH. feliscan inhibit the development of oral tolerance to OVA in mice and that this inhibition is prevented by rebamipide.
Purification and Characterization of an Immunogenic Aminopeptidase ofBrucella melitensis
Authors: Contreras-Rodriguez Araceli , Ramirez-Zavala Bernardo , Contreras Andrea , Schurig Gerhardt G. , Sriranganathan Nammalwar , Lopez-Merino Ahide
Published: August 2003

An immunogenic aminopeptidase was purified fromBrucella melitensisstrain VTRM1. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps. This procedure resulted in a yield of 29% and a 144-fold increase in specific activity. The aminopeptidase appeared to be a monomeric enzyme with a molecular mass of 96 kDa and an isoelectric point of 4.8. Its activity was optimal at pH 7.0 at 40°C. The enzyme was strongly inhibited by EDTA, 1,10-phenathroline, and divalent cations (Zn2+and Hg2+), suggesting that this protein was a metalloaminopeptidase. The enzyme showed preference for alanine at the N termini of aminoacyl derivatives. TheKmvalues forl-alanine-p-nitroanilide (Ala-pNA) and Lys-pNA were 0.35 and 0.18 mM, respectively. The N-terminal sequence of aminopeptidase was used for a homologous search in the genomes ofB. melitensis16M andBrucella suis1330. The analysis revealed an exact match of the probe sequence (36 bp) with an open reading frame of 2,652 bp encoding a protein predicted to be alanyl aminopeptidase (aminopeptidase N). Collectively, these data suggest designation of theB. melitensisenzyme as an aminopeptidase N. The aminopeptidase was recognized by sera from patients with acute and chronic brucellosis, suggesting that the enzyme may have important diagnostic implications.
Mannose-Binding Lectin Is a Disease Modifier in Clinical Malaria and May Function as Opsonin forPlasmodium falciparum- Infected Erythrocytes
Authors: Garred Peter , Nielsen Morten A. , Kurtzhals Jørgen A.L. , Malhotra Rajneesh , Madsen Hans O. , Goka Bamenla Q. , Akanmori Bartholomew D. , Sim Robert B. , Hviid Lars
Published: August 2003

Variant alleles in the mannose-binding lectin (MBL) gene (mbl2) causing low levels of functional MBL are associated with susceptibility to different infections and are common in areas where malaria is endemic. Therefore, we investigated whether MBL variant alleles in 551 children from Ghana were associated with the occurrence and outcome parameters ofPlasmodium falciparummalaria and asked whether MBL may function as an opsonin forP. falciparum. No difference in MBL genotype frequency was observed between infected and noninfected children or between children with cerebral malaria and/or severe malarial anemia and children with uncomplicated malaria. However, patients with complicated malaria who were homozygous for MBL variant alleles had significantly higher parasite counts and lower blood glucose levels than their MBL-competent counterparts. Distinct calcium-dependent binding of MBL to the membrane ofP. falciparum-infected erythrocytes, which could be inhibited by mannose, was observed. Further characterization revealed that MBL reacted with aP. falciparumglycoprotein identical to the 78-kDa glucose-regulated stress protein ofP. falciparum. MBL seems to be a disease modifier in clinical malaria and to function as an opsonin for erythrocytes invaded byP. falciparumand may thus be involved in sequestration of the parasite, which in turn may explain the association between homozygosity for MBL variant alleles and high parasite counts.
SWR Mice Are Highly Susceptible to Pulmonary Infection with
Mycobacterium tuberculosis
Authors: Turner Oliver C. , Keefe Robert G. , Sugawara Isamu , Yamada Hiroyuki , Orme Ian M.
Published: August 2003

Inbred mice differ in their abilities to control the growth of
Mycobacterium tuberculosis
in the lung and can as a result be regarded as either resistant or susceptible strains. In this study we report that the SWR mouse is both highly susceptible and in addition appears incapable of establishing a characteristic state of chronic disease after low-dose aerosol infection. In comparison to C57BL/6 mice, SWR mice were unable to contain the bacterial load in the lungs, resulting in progressive fatal disease. Histologic analysis of the lung tissue revealed evidence of a florid inflammatory cell response in the SWR mice leading to degeneration and necrosis and consolidation of a large percentage of the lung surface area. Digestion of infected lungs and analysis by flow cytometry demonstrated an initially similar but eventually higher number of lymphocytes accumulating in the SWR mice. Additionally, in contrast to the C57BL/6 mice, SWR mice had a significantly lower percentage of CD4 T cells in the lungs showing evidence of proliferation and positive intracellular staining for gamma interferon during the first two months of infection, and a lower percentage of both CD4 and CD8T cells exhibiting differentiation to an effector/memory phenotype during the first month of infection. We propose that further investigation of the SWR mouse may provide a new animal model for immunocompetent individuals apparently unable to effectively control the growth of
M. tuberculosis
in the lung.
Differential Role of MyD88 in Macrophage-Mediated Responses to Opportunistic Fungal Pathogens
Authors: Marr Kieren A. , Arunmozhi Balajee S. , Hawn Thomas R. , Ozinsky Adrian , Pham Uyenvy , Akira Shizuo , Aderem Alan , Conrad Liles W.
Published: August 2003

Toll-like receptors mediate macrophage recognition of microbial ligands, inducing expression of microbicidal molecules and cytokines via the adapter protein MyD88. We investigated the role of MyD88 in regulating murine macrophage responses to a pathogenic yeast (
Candida albicans
) and mold (
Aspergillus fumigatus
). Macrophages derived from bone marrow of MyD88-deficient mice (MyD88
−/−
) demonstrated impaired phagocytosis and intracellular killing of
C. albicans
compared to wild-type (MyD88
+/+
) macrophages. In contrast, ingestion and killing of
A. fumigatus
conidia was MyD88 independent. Cytokine production by MyD88
−/−
macrophages in response to
C. albicans
yeasts and hyphae was substantially decreased, but responses to
A. fumigatus
hyphae were preserved. These results provide evidence that MyD88 signaling is involved in phagocytosis and killing of live
C. albicans
, but not
A. fumigatus
. The differential role of MyD88 may represent one mechanism by which macrophages regulate innate responses specific to different pathogenic fungi.
Both the Fas Ligand and Inducible Nitric Oxide Synthase Are Needed for Control of Parasite Replication within Lesions in Mice Infected withLeishmania majorwhereas the Contribution of Tumor Necrosis Factor Is Minimal
Authors: Chakour Reza , Guler Reto , Bugnon Mélanie , Allenbach Cindy , Garcia Irène , Mauël Jacques , Louis Jacques , Tacchini-Cottier Fabienne
Published: August 2003

Following infection with the protozoan parasiteLeishmania major, C57BL/6 mice develop a small lesion that heals spontaneously. Resistance to infection is associated with the development of CD4+Th1 cells producing gamma interferon (IFN-γ) and tumor necrosis factor (TNF), which synergize in activating macrophages to their microbicidal state. We show here that C57BL/6 mice lacking both TNF and Fas ligand (FasL) (gldTNF−/−mice) infected withL. majorneither resolved their lesions nor controlledLeishmaniareplication despite the development of a strong Th1 response. Comparable inducible nitric oxide synthase (iNOS) activities were detected in lesions of TNF−/−,gldTNF−/−, andgldmice, but onlygldandgldTNF−/−mice failed to control parasite replication. Parasite numbers were high ingldmice and even more elevated ingldTNF−/−mice, suggesting that, in addition to iNOS, the Fas/FasL pathway is required for successful control of parasite replication and that TNF contributes only a small part to this process. Furthermore, FasL was shown to synergize with IFN-γ for the induction of leishmanicidal activity within macrophages infected withL. majorin vitro. Interestingly, TNF−/−mice maintained large lesion size throughout infection, despite being able to largely control parasite numbers. Thus, IFN-γ, FasL, and iNOS appear to be essential for the complete control of parasite replication, while the contribution of TNF is more important in controlling inflammation at the site of parasite inoculation.
Characterization of
Pseudomonas aeruginosa
Exoenzyme S as a Bifunctional Enzyme in J774A.1 Macrophages
Authors: Rocha Claudia L. , Coburn Jenifer , Rucks Elizabeth A. , Olson Joan C.
Published: August 2003

Pseudomonas aeruginosa
exoenzyme S (ExoS) is a type III secretion (TTS) effector, which includes both a GTPase-activating protein (GAP) activity toward the Rho family of low-molecular-weight G (LMWG) proteins and an ADP-ribosyltransferase (ADPRT) activity that targets LMWG proteins in the Ras, Rab, and Rho families. The coordinate function of both activities of ExoS in J774A.1 macrophages was assessed by using
P. aeruginosa
strains expressing and translocating wild-type ExoS or ExoS defective in GAP and/or ADPRT activity. Distinct and coordinated functions were identified for both domains. The GAP activity was required for the antiphagocytic effect of ExoS and was linked to interference of lamellopodium and membrane ruffle formation. Alternatively, the ADPRT activity of ExoS altered cellular adherence and morphology and was linked to effects on filopodium formation. The cellular mechanism of ExoS GAP activity included an inactivation of Rac1 function, as determined in p21-activated kinase 1-glutathione
S
-transferase (GST) pull-down assays. The ADPRT activity of ExoS targeted Ras and RalA but not Rab or Rho proteins, and Ral binding protein 1-GST pull-down assays identified an effect of ExoS ADPRT activity on RalA activation. The results from these studies confirm the bifunctional nature of ExoS activity within macrophages when translocated by TTS.
Ehrlichia chaffeensis
and
Anaplasma phagocytophilum
Lack Genes for Lipid A Biosynthesis and Incorporate Cholesterol for Their Survival
Authors: Lin Mingqun , Rikihisa Yasuko
Published: August 2003

Ehrlichia chaffeensis
and
Anaplasma phagocytophilum
are agents of human monocytic and granulocytic ehrlichioses, respectively. They are extremely sensitive to mechanical stress and are pleomorphic gram-negative bacteria. Membrane incorporation of cholesterol from the eukaryotic host is known to be essential for other fragile and pleomorphic bacteria and mycoplasmas that lack a cell wall. Thus, we tested whether cholesterol is required for
E. chaffeensis
and
A. phagocytophilum
. Using a freeze fracture technique and biochemical analysis, these bacteria were found to contain significant levels of membrane cholesterol. These bacteria lack genes for cholesterol biosynthesis or modification. However, host cell-free bacteria had the ability to take up directly exogenous cholesterol or NBD-cholesterol, a fluorescent cholesterol derivative. Treatment of the bacteria with cholesterol extraction reagent methyl-β-cyclodextrin caused their ultrastructural changes. Furthermore, pretreatment of the bacteria with methyl-β-cyclodextrin or NBD-cholesterol deprived these bacteria of the ability to infect leukocytes, thus killing these obligate intracellular bacteria. Analysis of
E. chaffeensis
and
A. phagocytophilum
genome sequences revealed that these bacteria lack all genes for the biosynthesis of lipid A and most genes for the biosynthesis of peptidoglycan, which confer structural strength to gram-negative bacteria. Taken together, these results suggest that human ehrlichiosis agents became cholesterol dependent due to the loss of these genes. As the first report of gram-negative bacteria incorporating cholesterol for survival, these findings offer insight into the unique nature of their parasitism and imply that cholesterol is important in the control of human ehrlichioses.