bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced Antigen-Specific Delayed-Type Hypersensitivity and Immunoglobulin G2b Responses after Oral Administration of ViableLactobacillus caseiYIT9029 in Wistar and Brown Norway Rats
Authors: de Waard R. , Garssen J. , Snel J. , Bokken G. C. A. M. , Sako T. , Huis in 't Veld J. H. J. , Vos J. G.
Published: July 2002

In this study, the effects of orally administered viableLactobacillus caseiShirota strain YIT9029 on the immunity parameters of Wistar and Brown Norway rats were examined. For this purpose, we used theTrichinella spiralishost resistance model. Two weeks before and duringT. spiralisinfection, rats were fed 109viableL. caseibacteria 5 days per week. TheT. spiralis-specific delayed-type hypersensitivity (DTH) response was significantly enhanced in both Wistar and Brown Norway rats givenL. casei. In both rat strains fedL. casei, serumT. spiralis-specific immunoglobulin G2b (IgG2b) concentrations were also significantly increased. In the model, no significant effects ofL. caseion larval counts or inflammatory reactions in the tongue musculature, body weights, or lymphoid organ weights were observed. Serum specific antibody responses, other than IgG2b, were not changed by feeding ofL. casei. In contrast toL. casei, it was shown that orally administeredBifidobacterium breveorBifidobacterium bifidumhad no influence on the measured infection and immunity indices in the rat infection model. Since the rat DTH response is considered to be a manifestation of Th1 cell-mediated immunity and the IgG2b isotype has been associated with Th1 activity, it was concluded that Th1 cells could play an active role in the immunomodulatory effects of orally administeredL. casei. Furthermore, our data do not indicate that the effect of oral supplementation withL. caseiis dependent on the genetic background of the host.
Evaluation of Two Enzyme-Linked Immunosorbent Assays for DetectingSalmonella entericasubsp.entericaSerovar Dublin Antibodies in Bulk Milk
Authors: Veling J. , van Zijderveld F. G. , van Zijderveld-van Bemmel A. M. , Schukken Y. H. , Barkema H. W.
Published: July 2002

Two enzyme-linked immunosorbent assays (ELISAs) for the detectingSalmonella entericasubsp. entericaserovar Dublin antibodies in bulk milk were developed and evaluated for potential use in control programs. The ELISAs were based on either lipopolysacharide (LPS ELISA) or flagellar antigen (GP ELISA). Sensitivity was determined with 79 case herds with a wide range of clinical signs. Specificity was determined with 125 Dutch and 200 Swedish control herds. The relation between antibodies in bulk milk, antibodies in serum, and the level of milk production of individual cows was studied with 61 case herds. The optimal optical density (OD) values of the LPS ELISA and the GP ELISA were determined to be 0.2 and 0.5, respectively. The sensitivities of the LPS ELISA and the GP ELISA were 54 and 63%, respectively, with a specificity of 98% for both ELISAs with samples from the Dutch control herds. The specificities for samples from the Swedish herds were 100% for the LPS ELISA and 95% for the GP ELISA. The sensitivity of the combination of tests was 65% when samples were run in parallel, and the specificity was 100% when samples were run in series, irrespective of whether the samples came from Dutch or Swedish control herds. The variance (R2) in the OD value for bulk milk samples could be explained by the percentage of seropositive lactating cows in a herd with the LPS ELISA for 51% of the samples and with the GP ELISA for 72%. The variance in the OD value was best explained by the combination of the percentage of seropositive lactating cows in the herd and the mean log10serum antibody titer for that herd (R2= 62% for the LPS ELISA andR2= 75% for the GP ELISA). Case herds more often tested negative by the ELISA with bulk milk when the percentage of seropositive lactating cows was less than 5%. It is concluded that both ELISAs with bulk milk can be used in control programs to distinguish between infected and noninfected herds. Specificity can be increased by using the two tests in combination. Sensitivity was relatively low for both single tests and both tests combined.
Diagnosis of Noncultivatable Gastroenteritis Viruses, the Human Caliciviruses
Authors: Atmar Robert L. , Estes Mary K.
Published: July 2002

Gastroenteritis is one of the most common illnesses of humans, and many different viruses have been causally associated with this disease. Of those enteric viruses that have been established as etiologic agents of gastroenteritis, only the human caliciviruses cannot be cultivated in vitro. The cloning of Norwalk virus and subsequently of other human caliciviruses has led to the development of several new diagnostic assays. Antigen detection enzyme immunoassays (EIAs) using polyclonal hyperimmune animal sera and antibody detection EIAs using recombinant virus-like particles have supplanted the use of human-derived reagents, but the use of these assays has been restricted to research laboratories. Reverse transcription-PCR assays for the detection of human caliciviruses are more widely available, and these assays have been used to identify virus in clinical specimens as well as in food, water, and other environmental samples. The application of these newer assays has significantly increased the recognition of the importance of human caliciviruses as causes of sporadic and outbreak-associated gastroenteritis.
αvβ3Integrin and Bacterial Lipopolysaccharide Are Involved inCoxiella burnetii-Stimulated Production of Tumor Necrosis Factor by Human Monocytes
Authors: Dellacasagrande Jérôme , Ghigo Eric , Machergui-El Sarah , Hammami  , Toman Rudolf , Raoult Didier , Capo Christian , Mege Jean-Louis
Published: July 2002

Coxiella burnetii, the agent of Q fever, enters human monocytes through αvβ3integrin and survives inside host cells. In addition,C. burnetiistimulates the synthesis of inflammatory cytokines including tumor necrosis factor (TNF) by monocytes. We studied the role of the interaction ofC. burnetiiwith THP-1 monocytes in TNF production. TNF transcripts and TNF release reached maximum values within 4 h. Almost all monocytes boundC. burnetiiafter 4 h, while the percentage of phagocytosing monocytes did not exceed 20%. Cytochalasin D, which prevented the uptake ofC. burnetiiwithout interfering with its binding, did not affect the expression of TNF mRNA. Thus, bacterial adherence, but not phagocytosis, is necessary for TNF production by monocytes. The monocyte αvβ3integrin was involved in TNF synthesis since peptides containing RGD sequences and blocking antibodies against αvβ3integrin inhibited TNF transcripts induced byC. burnetii. Nevertheless, the cross-linking of αvβ3integrin by specific antibodies was not sufficient to induce TNF synthesis. The signal delivered byC. burnetiiwas triggered by bacterial lipopolysaccharide (LPS). Polymyxin B inhibited the TNF production stimulated byC. burnetii, and soluble LPS isolated fromC. burnetiilargely mimicked viable bacteria. On the other hand, avirulent variants ofC. burnetiiinduced TNF production through an increased binding to monocytes rather than through the potency of their LPS. We suggest that the adherence ofC. burnetiito monocytes via αvβ3integrin enables surface LPS to stimulate TNF production in THP-1 monocytes.
Analysis of Cytadherence-Deficient, GapA-Negative
Mycoplasma gallisepticum
Strain R
Authors: Papazisi L. , Troy K. E. , Gorton T. S. , Liao X. , Geary S. J.
Published: July 2002

Comparison of the phenotypic expression of
Mycoplasma gallisepticum
strain R low (passage 15) to that of strain R high (passage 164) revealed that three proteins, i.e., the cytadhesin molecule GapA, a 116-kDa protein (p116), and a 45-kDa protein (p45), are missing in strain R high. Sequence analysis confirmed that the insertion of an adenine 105 bp downstream of the
gapA
translational start codon resulted in premature termination of translation in R high. A second adenine insertion had also occurred at position 907. Restoration of expression of wild-type
gapA
in R high (clone designated GT5) allowed us to evaluate the extent to which the diminished cytadherence capacity could be attributed to GapA alone. The results indicated that GT5 attached to the same limited extent as the parental R high, from which it was derived. The cytadherence capability of the parental R high was not restored solely by
gapA
complementation alone, indicating that either p116 or p45 or both may play a role in the overall cytadherence process. The gene encoding p116 was found to be immediately downstream of
gapA
in the same operon and was designated
crmA
. This gene exhibited striking homology to genes encoding molecules with cytadhesin-related functions in both
Mycoplasma pneumoniae
and
Mycoplasma genitalium
. Transcriptional analysis revealed that
crmA
is not transcribed in R high. We are currently constructing a shuttle vector containing both the wild-type
gapA
and
crmA
for transformation into R high to assess the role of CrmA in the cytadherence process.
Cytotoxic Activity of Coagulase-Negative Staphylococci in Bovine Mastitis
Authors: Zhang Songlin , Maddox Carol W.
Published: July 2002

Secreted toxins play important roles in the pathogenesis of bacterial infections. In this study, we examined the presence of secreted cytotoxic factors of coagulase-negative staphylococci (CoNS) from bovine clinical and subclinical mastitis. A 34- to 36-kDa protein with cell-rounding cytotoxic activity was found in many CoNS strains, especially inStaphylococcus chromogenesstrains. The protein caused cell detachment and cell rounding in several cell lines, including HEp-2, Int 407, CHO-K1, and Y-1 cells. Native protein recovered from nondenatured polyacrylamide gel electrophoresis showed both cytotoxic activity and casein hydrolysis activity. The purified protein had a pH optimal at 7.2 to 7.5 and a pI of 5.1 and was heat labile. The proteolytic activity could be inhibited by zinc and metal specific inhibitors such as 1,10-phenanthroline and EDTA, indicating that it is a metalloprotease. Protein mass analysis and peptide sequencing indicated that the protein is a novel metalloprotease. Different bacterial strains expressed variable levels of 34- to 36-kDa protease, which may provide an indication of strain virulence.
Synthetic Peptide Immunogens Elicit Polyclonal and Monoclonal Antibodies Specific for Linear Epitopes in the D Motifs of
Staphylococcus aureus
Fibronectin-Binding Protein, Which Are Composed of Amino Acids That Are Essential for Fibronectin Binding
Authors: Huesca Mario , Sun Qing , Peralta Robert , Shivji Gulnar M. , Sauder Daniel N. , McGavin Martin J.
Published: July 2002

A fibronectin (Fn)-binding adhesin of
Staphylococcus aureus
contains three tandem 37- or 38-amino-acid motifs (D1, D2, and D3), which function to bind Fn. Plasma from patients with
S. aureus
infections contain antibodies that preferentially recognize ligand induced binding sites in the D motifs and do not inhibit Fn binding (F. Casolini, L. Visai, D. Joh, P. G. Conaldi, A. Toniolo, M. Höök, and P. Speziale, Infect. Immun. 66:5433–5442, 1998). To eliminate the influence of Fn binding on antibody development, we used synthetic peptide immunogens D1
21–34
and D3
20–33
, which each contain a conserved pattern of amino acids that is essential for Fn binding but which cannot bind Fn without N- or C-terminal extensions. The D3
20–33
immunogen promoted the production of polyclonal antibodies that were 10-fold more effective as inhibitors of Fn-binding to the D3 motif than antibodies obtained by immunizing with an extended peptide D3
16–36
, which exhibits functional Fn binding. The D3
20–33
immunogen also facilitated the production of a monoclonal antibody, 9C3, which was highly specific for the epitope SVDFEED, and abolished Fn binding by the D3 motif. When mixed with polyclonal anti-D1
21–34
immunoglobulin G, 70% inhibition of Fn binding to the three tandem D motifs was achieved compared to no more than 30% inhibition with either antibody preparation alone. Therefore, by immunizing with short synthetic peptides that are unable to bind Fn, we have effectively stimulated the production of antibodies specific for epitopes comprised of amino acids that are essential for Fn binding. Although these epitopes occur within a conserved pattern of amino acids that is required for Fn binding, the antibodies recognized specific linear epitope sequences and not a conserved structure common to all repeated motifs.
Lipopolysaccharide Entry in the Damaged Cornea and Specific Uptake by Polymorphonuclear Neutrophils
Authors: Schultz C. L. , Buret A. G. , Olson M. E. , Ceri H. , Read R. R. , Morck D. W.
Published: July 2002

Bacterial lipopolysaccharide (LPS) is an important agent of induction of ocular pathology following corneal injury or wearing of contaminated contact lenses. The mechanism of LPS uptake through the corneal epithelium is unclear, and the role played by inflammatory cells in this phenomenon has not been previously assessed. Fluorescein isothiocyanate-labeled LPS from
Escherichia coli
was deposited onto the abraded corneas of New Zealand White rabbits. Epifluorescence microscopy of living excised corneas revealed diffuse LPS staining in the epithelial and stromal layers only in the vicinity of the abrasion. In addition, specific cellular uptake of LPS was suggested by fluorescence staining of cells along the abrasion site. In a second series of experiments, an anti-CD18 polyclonal antibody was used to block infiltration of polymorphonuclear neutrophils (PMN) into the cornea. In these experiments, a diffuse distribution of fluorescent LPS was still observed along the abrasion, but the specific cellular uptake was abolished. The findings indicate that LPS enters the cornea via diffuse penetration at sites of injury and that specific cellular uptake of LPS occurs within the cornea via PMN which have migrated into the damaged tissue.
Immunoglobulin G3 Antibodies Specific for the 19-Kilodalton Carboxyl-Terminal Fragment ofPlasmodium yoeliiMerozoite Surface Protein 1 Transfer Protection to Mice Deficient in Fc-γRI Receptors
Authors: Vukovic Peter , Hogarth P. Mark , Barnes Nadine , Kaslow David C. , Good Michael F.
Published: July 2002

Merozoite surface protein 1 (MSP-119) is a leading malaria vaccine candidate. Specific antibodies contribute to immunity; binding to macrophages is believed to represent the main action of malaria antibodies. We show that an MSP-119-specific immunoglobulin G3 (IgG3) monoclonal antibody can passively transfer protection to mice deficient in the α chain of Fc-γRI whose macrophages cannot bind IgG3.
Transcriptional Organization and Function of Invasion Genes within
Salmonella enterica
Serovar Typhimurium Pathogenicity Island 1, Including the
prgH
,
prgI
,
prgJ
,
prgK
,
orgA
,
orgB
, and
orgC
Genes
Authors: Klein Joanna R. , Fahlen Thomas F. , Jones Bradley D.
Published: July 2002

Salmonella enterica
serovar Typhimurium initiates infection of a host by inducing its own uptake into specialized M cells which reside within the epithelium overlaying Peyer's patches. Entry of
Salmonella
into intestinal epithelial cells is dependent upon invasion genes that are clustered together in
Salmonella
pathogenicity island 1 (SPI-1). Upon contact between serovar Typhimurium and epithelial cells targeted for bacterial internalization, bacterial proteins are injected into the host cell through a type III secretion system that leads to internalization of the bacteria. Previous work has established that the
prgH
, -
I
, -
J
, and -
K
and
orgA
genes reside in SPI-1, and the products of these genes are predicted to be components of the invasion secretion apparatus. We report that an error in the published
orgA
DNA sequence has been identified so that this region encodes two small genes rather than a single large open reading frame. These genes have been designated
orgA
and
orgB
. Additionally, an opening reading frame downstream of
orgB
, which we have designated
orgC
, has been identified and partially characterized. Previously published work has indicated that the
prgH
, -
I
, -
J
, and -
K
genes are transcribed from a promoter distinct from that used by the gene immediately downstream,
orgA
. Here, we present experiments indicating that
orgA
expression is driven by the
prgH
promoter. In addition, using reverse transcriptase PCR analysis, we have found that this polycistronic message extends downstream of
prgH
to include a total of 10 genes. To more fully characterize this invasion operon, we demonstrate that the
prgH
,
prgI
,
prgJ
,
prgK
,
orgA
, and
orgB
genes are each required for invasion and secretion, while
orgC
is not essential for the invasive phenotype.
The Capsule Is a Virulence Determinant in the Pathogenesis ofPasteurella multocidaM1404 (B:2)
Authors: Boyce John D. , Adler Ben
Published: July 2002

Capsules from a range of pathogenic bacteria are key virulence determinants, and the capsule has been implicated in virulence inPasteurella multocida. We have previously identified and determined the nucleotide sequence of theP. multocidaM1404 (B:2) capsule biosynthetic locus (J. D. Boyce, J. Y. Chung, and B. Adler, Vet. Microbiol. 72:121–134, 2000). Thecaplocus consists of 15 genes, which can be grouped into three functional regions. Regions 1 and 3 contain genes proposed to encode proteins involved in capsule export, and region 2 contains genes proposed to encode proteins involved in polysaccharide biosynthesis. In order to construct a mutant impaired in capsule export, the final gene of region 1,cexA, was disrupted by insertion of a tetracycline resistance cassette by allelic replacement. The genotype of thetet(M) ΩcexAmutant was confirmed by Southern hybridization and PCR. The acapsular phenotype was confirmed by immunofluorescence, and the strain could be complemented and returned to capsule production by the presence of a cloned uninterrupted copy ofcexA. Wild-type, mutant, and complemented strains were tested for virulence by intraperitoneal challenge of mice; the presence of the capsule was shown to be a crucial virulence determinant. Following intraperitoneal challenge of mice, the acapsular bacteria were removed efficiently from the blood, spleen, and liver, while wild-type bacteria multiplied rapidly. Acapsular bacteria were readily taken up by murine peritoneal macrophages, but wild-type bacteria were significantly resistant to phagocytosis. Both wild-type and acapsular bacteria were resistant to complement in bovine and murine serum.
Detection of Phase Variation in Expression of Proteins Involved in Hemoglobin and Hemoglobin-Haptoglobin Binding by Nontypeable
Haemophilus influenzae
Authors: Cope Leslie D. , Hrkal Zbynek , Hansen Eric J.
Published: July 2002

Haemophilus influenzae
can utilize different protein-bound forms of heme for growth in vitro. A previous study (I. Maciver, J. L. Latimer, H. H. Liem, U. Muller-Eberhard, Z. Hrkal, and E. J. Hansen. Infect. Immun. 64:3703–3712, 1996) indicated that nontypeable
H. influenzae
(NTHI) strain TN106 expressed a protein that bound hemoglobin-haptoglobin and was encoded by an open reading frame (ORF) that contained a CCAA nucleotide repeat. Southern blot analysis revealed that several NTHI strains contained between three and five chromosomal DNA fragments that bound an oligonucleotide probe for CCAA repeats. Three ORFs containing CCAA repeats were identified in NTHI strain N182; two of these ORFs were arranged in tandem. The use of translational fusions involving these three ORFs and the β-lactamase gene from pBR322 revealed that these three ORFs, designated
hgbA
,
hgbB
, and
hgbC
, encoded proteins that could bind hemoglobin, hemoglobin-haptoglobin, or both compounds. Monoclonal antibodies (MAbs) specific for the HgbA, HgbB, and HgbC proteins were produced by immunizing mice with synthetic peptides unique to each protein. Both HgbA and HgbB were readily detected by Western blot analysis in N182 cells grown in the presence of hemoglobin as the sole source of heme, whereas expression of HgbC was found to be much less abundant than that of HgbA and HgbB. The use of these MAbs in a colony blot radioimmunoassay analysis revealed that expression of both HgbA and HgbB was subject to phase variation. PCR and nucleotide sequence analysis were used in conjunction with Western blot analyses to demonstrate that this phase variation involved the CCAA repeats in the
hgbA
and
hgbB
ORFs.
Up-Regulation of Both Intimin and
eae
-Independent Adherence of Shiga Toxigenic
Escherichia coli
O157 by
ler
and Phenotypic Impact of a Naturally Occurring
ler
Mutation
Authors: Ogierman Monica A. , Paton Adrienne W. , Paton James C.
Published: July 2002

Shiga toxigenic
Escherichia coli
(STEC) strains are important human pathogens which are capable of causing diarrhea, hemorrhagic colitis, and the potentially fatal hemolytic-uremic syndrome (HUS). An important virulence trait of certain STEC strains, such as those belonging to serogroup O157, is the capacity to produce attaching and effacing (A/E) lesions on enterocytes, a property encoded by the locus for enterocyte effacement (LEE). LEE contains the
eae
gene, which encodes intimin, an outer membrane protein which mediates the intimate attachment of bacteria to the host epithelial cell surface, and
eae
is routinely used as a marker for LEE-positive STEC strains. However, the O157:H
−
STEC strain 95SF2 carries
eae
but did not produce A/E lesions on HEp-2 cells, as judged by a fluorescent actin staining assay. In this assay, 95SF2 adhered poorly to the HEp-2 cells, and those that did bind exhibited abnormal cell division. In contrast, the O157:H7 STEC strain EDL933 adhered strongly and produced typical A/E lesions. We have demonstrated that 95SF2 carries a defective LEE regulatory gene,
ler
, with a single base change with respect to that published for
ler
of EDL933, resulting in an Ile
57
-to-Thr substitution. Ler shows homology to H-NS-like regulators, which are modulators of transcription, and the mutation occurs in a domain implicated in oligomerization. 95SF2 was able to adhere and produce A/E lesions on HEp-2 cells when EDL933
ler
was expressed from a multicopy plasmid. Conversely, introduction of a plasmid carrying 95SF2
ler
into EDL933 abolished adherence and capacity to form A/E lesions. Studies with
eae
deletion derivatives of 95SF2 and EDL933 demonstrated that the
ler
-mediated adherence to HEp-2 cells is largely independent of intimin. We have also demonstrated that EDL933
ler
, but not 95SF2
ler
, increases the level of intimin in O157 STEC.
Amino Acid Transport into Cultured McCoy Cells Infected with
Chlamydia trachomatis
Authors: Harper Angela , Pogson Christopher I. , Pearce John H.
Published: July 2002

Amino acid transport into McCoy cells infected with strains representative of the two major biovars of
Chlamydia trachomatis
has been studied to determine if uptake is increased during infection. Preliminary work suggested that the transport systems L, A/ASC (for neutral amino acid transport), N (for transport of Asn, Gln, and His) and y+ (for cationic amino acids) were present in McCoy cells. With lymphogranuloma venereum biovar strain 434, little difference in the influx of representative amino acids Trp, His, and Lys or the analogue 2-aminoisobutyric acid (AIB) was observed during infection. With trachoma biovar strain DK20, a small increase in the initial entry rate and equilibrium concentration of each amino acid was found. McCoy cells appear to have great capacity for concentrating amino acids, which might obviate the need for transport induction by chlamydiae under conditions favoring the growth of infectious organisms.
Role of Plasma, Lipopolysaccharide-Binding Protein, and CD14 in Response of Mouse Peritoneal Exudate Macrophages to Endotoxin
Authors: Heumann Didier , Adachi Yoshiyuki , Le Roy Didier , Ohno Naohito , Yadomae Toshiro , Glauser Michel Pierre , Calandra Thierry
Published: July 2002

Plasma lipopolysaccharide (LPS)-binding protein (LBP) and membrane CD14 function to enhance the responses of monocytes to low concentrations of endotoxin. Surprisingly, recent reports have suggested that LBP or CD14 may be dispensable for macrophage responses to low concentrations of LPS or may even exert an inhibitory effect in the case of LBP. We therefore investigated whether LBP and CD14 participated in the response of mouse peritoneal exudate macrophages (PEM) to LPS stimulation. In the presence of a low amount of plasma (<1%) or of recombinant mouse or human LBP, PEM were found to respond to low concentrations of LPS (<5 to 10 ng/ml) in an LBP- and CD14-dependent manner. However, tumor necrosis factor production (not interleukin-6 production) by LPS-stimulated PEM was reduced when cells were stimulated in the presence of higher concentrations of plasma or serum (5 or 10%). Yet, the inhibitory effect of plasma or serum was not mediated by LBP. Taken together with previous results obtained withLBPandCD14knockout mice in models of experimental endotoxemia, the present data confirm a critical part for LBP and CD14 in innate immune responses of both blood monocytes and tissue macrophages to endotoxins.
Tir Tyrosine Phosphorylation and Pedestal Formation Are Delayed in Enteropathogenic
Escherichia coli sepZ
::Tn
phoA
Mutant 30-5-1(3)
Authors: Devinney Rebekah , Nisan Israel , Ruschkowski Sharon , Rosenshine Ilan , Finlay B. Brett
Published: July 2002

Enteropathogenic
Escherichia coli
(EPEC) strain 30-5-1(3) has been reported to form attaching and effacing (A/E) lesions without Tir tyrosine phosphorylation. In this study, we show that 30-5-1(3), which has a transposon insertion within the
sepZ
gene, forms wild-type A/E lesions including Tir tyrosine phosphorylation, but at a slower rate. A/E lesion formation by 30-5-1(3) occurs without detectable secretion of Tir or other EPEC Esp secreted proteins.
Mannheimia haemolyticaLeukotoxin Activates a Nonreceptor Tyrosine Kinase Signaling Cascade in Bovine Leukocytes, Which Induces Biological Effects
Authors: Jeyaseelan S. , Kannan M. S. , Briggs R. E. , Thumbikat P. , Maheswaran S. K.
Published: July 2002

The leukotoxin (LktA) produced byMannheimia haemolyticabinds to bovine lymphocyte function-associated antigen 1 (LFA-1) and induces biological effects in bovine leukocytes in a cellular and species-specific fashion. We have previously shown that LktA also binds to porcine LFA-1 without eliciting any effects. These findings suggest that the specificity of LktA effects must entail both binding to LFA-1 and activation of signaling pathways which are present in bovine leukocytes. However, the signaling pathways leading to biological effects upon LktA binding to LFA-1 have not been characterized. In this context, several reports have indicated that ligand binding to LFA-1 results in activation of a nonreceptor tyrosine kinase (NRTK) signaling cascade. We designed experiments with the following objectives: (i) to determine whether LktA binding to LFA-1 leads to activation of NRTKs, (ii) to examine whether LktA-induced NRTK activation is target cell specific, and (iii) to determine whether LktA-induced NRTK activation is required for biological effects. We used a biologically inactive mutant leukotoxin (ΔLktA) for comparison with LktA. Our results indicate that LktA induces tyrosine phosphorylation (TP) of the CD18 tail of LFA-1 in bovine leukocytes. The ΔLktA mutant does not induce TP of the CD18 tail, albeit binding to bovine LFA-1. LktA-induced TP of the CD18 tail was attenuated by an NRTK inhibitor, herbimycin A; a phosphatidylinositol 3′-kinase (PI 3-kinase) inhibitor, wortmannin; and a Src kinase inhibitor, PP2, in a concentration-dependent manner. Furthermore, LktA induces TP of the CD18 tail in bovine, but not porcine, leukocytes. Moreover, LktA-induced intracellular calcium ([Ca2+]i) elevation was also inhibited by herbimycin A, wortmannin, and PP2. Thus, our data represent the first evidence that binding of LktA to bovine LFA-1 induces a species-specific NRTK signaling cascade involving PI 3-kinase and Src kinases and that this signaling cascade is required for LktA-induced biological effects.
Immunization with Components of Two Iron Uptake ABC Transporters Protects Mice against Systemic
Streptococcus pneumoniae
Infection
Authors: Brown Jeremy S. , Ogunniyi A. David , Woodrow Matthew C. , Holden David W. , Paton James C.
Published: July 2002

There has been considerable recent research into protein based
Streptococcus pneumoniae
vaccines as alternatives to the existing capsular antigen vaccines. PiuA and PiaA (formerly Pit1A and Pit2A) are recently identified lipoprotein components of
S. pneumoniae
iron uptake ABC transporters which are required for full virulence and are likely to be expressed on the surface of the bacterial cell membrane. We investigated the efficacy of recombinant PiuA and PiaA proteins at eliciting protective immunity in mice against systemic infection with
S. pneumoniae
. Both recombinant PiuA and PiaA generated antibody responses that cross-reacted with each other but not with pneumolysin and reacted with identical proteins from nine different
S. pneumoniae
serotypes. Mice immunized with recombinant PiuA and PiaA were protected against systemic challenge to a degree similar to those immunized with an existing protein vaccine candidate, PdB (a genetically modified pneumolysin toxoid). Immunization with a combination of both PiuA and PiaA resulted in additive protection and was highly protective against systemic infection with
S. pneumoniae
. PiuA and PiaA are therefore promising additional candidates for a novel
S. pneumoniae
vaccine using protein antigens.
Regulated Expression and Effect of Galectin-1 onTrypanosoma cruzi-Infected Macrophages: Modulation of Microbicidal Activity and Survival
Authors: Zúñiga Elina , Gruppi Adriana , Hirabayashi Jun , Kasai Ken I. , Rabinovich Gabriel A.
Published: July 2002

Galectin-1 is a β-galactoside-binding protein with potent anti-inflammatory and immunoregulatory effects. However, its expression and function have not been assessed in the context of an infectious disease. The present study documents, for the first time, the regulated expression of galectin-1 in the context of an infectious process and its influence in the modulation of macrophage microbicidal activity and survival. A biphasic modulation in parasite replication and cell viability was observed when macrophages isolated fromTrypanosoma cruzi-infected mice were exposed to increasing concentrations of galectin-1. While low concentrations of this protein increased parasite replication and did not affect macrophage survival, higher inflammatory doses of galectin-1 were able to commit cells to apoptosis and inhibited parasite replication. Furthermore, galectin-1 at its lowest concentration was able to down-regulate critical mediators for parasite killing, such as interleukin 12 (IL-12) and nitric oxide, while it did not affect IL-10 secretion. Finally, endogenous galectin-1 was found to be up-regulated and secreted by the J774 macrophage cell line cultured in the presence of trypomastigotes. This result was extended in vivo by Western blot analysis, flow cytometry, and reverse transcription-PCR using macrophages isolated fromT. cruzi-infected mice. This study documents the first association between galectin-1's immunoregulatory properties and its role in infection and provides new clues to the understanding of the mechanisms implicated in host-parasite interactions during Chagas' disease and other parasite infections.
Effect of Deficiency of Tumor Necrosis Factor Alpha or Both of Its Receptors onStreptococcus pneumoniaeCentral Nervous System Infection and Peritonitis
Authors: Wellmer Andreas , Gerber Joachim , Ragheb Jasmin , Zysk Gregor , Kunst Tammo , Smirnov Alexander , Brück Wolfgang , Nau Roland
Published: July 2002

Tumor necrosis factor alpha (TNF-α) and TNF-β are key mediators in bacterial inflammation. We therefore examined the role of TNF-α and its two receptors in murine pneumococcal central nervous system infection. TNF-α knockout mice and age- and sex-matched controls and TNF receptor (p55 and p75)-deficient mice and heterozygous littermates were infected intracerebrally with aStreptococcus pneumoniaetype 3 strain. Mice were monitored until death or were killed 36 h after infection. Bacterial titers in blood, spleen, and brain homogenates were determined. Leukocyte infiltration and neuronal damage were assessed by histological scores. TNF-α-deficient mice died earlier than the controls after intracerebral infection although overall survival was similar. TNF-α deficiency did not inhibit leukocyte recruitment into the subarachnoid space and did not lead to an increased density of bacteria in brain homogenates. However, it caused a substantial rise of the concentration ofS. pneumoniaecells in blood and spleen. Spleen bacterial titers were also increased in p55- and p75-deficient mice. TNF receptor-deficient mice showed decreased meningeal inflammation. Neuronal damage was not affected by either TNF-α or TNF receptor deficiency. In a murine model of pneumococcal peritonitis, 102CFU ofS. pneumoniaeproduced fatal peritonitis in TNF-α-deficient, but not wild-type, mice. Early leukocyte influx into the peritoneum was impaired in TNF-α-deficient mice. The lack of TNF-α or its receptors renders mice more susceptible toS. pneumoniaeinfections.
Characterization of Receptor-Mediated Signal Transduction byEscherichia coliType IIa Heat-Labile Enterotoxin in the Polarized Human Intestinal Cell Line T84
Authors: Wimer-Mackin Susan , Holmes Randall K. , Wolf Anne A. , Lencer Wayne I. , Jobling Michael G.
Published: July 2002

Escherichia colitype IIa heat-labile enterotoxin (LTIIa) binds in vitro with highest affinity to ganglioside GD1b. It also binds in vitro with lower affinity to several other oligosialogangliosides and to ganglioside GM1, the functional receptor for cholera toxin (CT). In the present study, we characterized receptor-mediated signal transduction by LTIIa in the cultured T84 cell model of human intestinal epithelium. Wild-type LTIIa bound tightly to the apical surface of polarized T84 cell monolayers and elicited a Cl−secretory response. LTIIa activity, unlike CT activity, was not blocked by the B subunit of CT. Furthermore, an LTIIa variant with a T14I substitution in its B subunit, which binds in vitro to ganglioside GM1 but not to ganglioside GD1b, was unable to bind to intact T84 cells and did not elicit a Cl−secretory response. These findings show that ganglioside GM1 on T84 cells is not a functional receptor for LTIIa. The LTIIa receptor on T84 cells was inactivated by treatment with neuraminidase. Furthermore, LTIIa binding was blocked by tetanus toxin C fragment, which binds to gangliosides GD1b and GT1b. These findings support the hypothesis that ganglioside GD1b, or possibly a glycoconjugate with a GD1b-like oligosaccharide, is the functional receptor for LTIIa on T84 cells. The LTIIa-receptor complexes from T84 cells were associated with detergent-insoluble membrane microdomains (lipid rafts), extending the correlation between toxin binding to lipid rafts and toxin function that was previously established for CT. However, the extent of association with lipid rafts and the magnitude of the Cl−secretory response in T84 cells were less for LTIIa than for CT. These properties of LTIIa and the previous finding that enterotoxin LTIIb binds to T84 cells but does not associate with lipid rafts or elicit a Cl−secretory response may explain the low pathogenicity for humans of type II enterotoxin-producing isolates ofE. coli.
Effect of 6-Hydroxydopamine on Host Resistance againstListeria monocytogenesInfection
Authors: Miura Tomisato , Kudo Tsuyoshi , Matsuki Akitomo , Sekikawa Kenji , Tagawa Yoh-Ichi , Iwakura Yoichiro , Nakane Akio
Published: July 2002

Recent studies have shown that immunocompetent cells bear receptors of neuropeptides and neurotransmitters and that these ligands play roles in the immune response. In this study, the role of the sympathetic nervous system in host resistance againstListeria monocytogenesinfection was investigated in mice pretreated with 6-hydroxydopamine (6-OHDA), which destroys sympathetic nerve termini. The norepinephrine contents of the plasma and spleens were significantly lower in 6-OHDA-treated mice than in vehicle-treated mice. The 50% lethal dose ofL. monocytogeneswas about 20 times higher for 6-OHDA-treated mice than for vehicle-treated mice. Chemical sympathectomy by 6-OHDA upregulated interleukin-12 (IL-12) and tumor necrosis factor-alpha (TNF-α) production in enriched dendritic cell cultures and gamma interferon (IFN-γ) and TNF-α production in spleen cell cultures, whereas chemical sympathectomy had no apparent effect on phagocytic activities, listericidal activities, and nitric oxide production in peritoneal exudate cells and splenic macrophages. Augmentation of host resistance againstL. monocytogenesinfection by 6-OHDA was abrogated in IFN-γ−/−or TNF-α−/−mice, suggesting that upregulation of IFN-γ, IL-12, and TNF-α production may be involved in 6-OHDA-mediated augmentation of antilisterial resistance. Furthermore, adoptive transfer of spleen cells immune toL. monocytogenesfrom 6-OHDA-treated mice resulted in untreated naive recipients that had a high level of resistance againstL. monocytogenesinfection. These results suggest that the sympathetic nervous system may modulate host resistance againstL. monocytogenesinfection through regulation of production of IFN-γ, IL-12, and TNF-α, which are critical in antilisterial resistance.
Role of Capsule in the Pathogenesis of Fowl Cholera Caused byPasteurella multocidaSerogroup A
Authors: Chung Jing Yeng , Wilkie Ian , Boyce John D. , Townsend Kirsty M. , Frost Alan J. , Ghoddusi Majid , Adler Ben
Published: July 2002

We have constructed a defined acapsular mutant inPasteurella multocidaX-73 (serogroup A:1) by disrupting thehexAgene through the insertion of a tetracycline resistance cassette. The genotype of thehexA::tet(M) strain was confirmed by PCR and Southern hybridization, and the acapsular phenotype of this strain was confirmed by electron microscopy. ThehexA::tet(M) strain was attenuated in both mice and chickens. Complementation of the mutant with an intacthexABfragment restored lethality in mice but not in chickens. In contrast to the results described previously forP. multocidaserogroup B (J. D. Boyce and B. Adler, Infect. Immun. 68:3463–3468, 2000), thehexA::tet(M) strain was sensitive to the bactericidal action of chicken serum, whereas the wild-type and complemented strains were both resistant. Following inoculation into chicken muscle, the bacterial count of thehexA::tet(M) strain decreased significantly, while the wild-type and complemented strains both grew rapidly over 4 h. The capsule is thus an essential virulence determinant in the pathogenesis of fowl cholera.
Mycobacterium bovis
BCG
recA
Deletion Mutant Shows Increased Susceptibility to DNA-Damaging Agents but Wild-Type Survival in a Mouse Infection Model
Authors: Sander Peter , Papavinasasundaram K. G. , Dick Thomas , Stavropoulos Evangelos , Ellrott Kerstin , Springer Burkhard , Colston M. Joseph , Böttger Erik C.
Published: July 2002

Pathogenic microorganisms possess antioxidant defense mechanisms for protection from reactive oxygen metabolites which are generated during the respiratory burst of phagocytic cells. These defense mechanisms include enzymes such as catalase, which detoxifies reactive oxygen species, and DNA repair systems, which repair damage resulting from oxidative stress. To (i) determine the relative importance of the DNA repair system when oxidative stress is encountered by the
Mycobacterium tuberculosis
complex during infection of the host and to (ii) provide improved mycobacterial hosts as live carriers to express foreign antigens, the
recA
locus was inactivated by allelic exchange in
Mycobacterium bovis
BCG. The
recA
mutants are sensitive to DNA-damaging agents and show increased susceptibility to metronidazole, the first lead compound active against the dormant
M. tuberculosis
complex. Surprisingly, the
recA
genotype does not affect the in vitro dormancy response, nor does the defect in the DNA repair system lead to attenuation as determined in a mouse infection model. The
recA
mutants will be a valuable tool for further development of BCG as an antigen delivery system to express foreign antigens and as a source of a genetically stable vaccine against tuberculosis.
Genotypic Variation in the
Bordetella pertussis
Virulence Factors Pertactin and Pertussis Toxin in Historical and Recent Clinical Isolates in the United Kingdom
Authors: Fry Norman K. , Neal Shona , Harrison Timothy G. , Miller Elizabeth , Matthews Ruth , George Robert C.
Published: July 2002

The reemergence of pertussis has been reported in several countries despite high vaccination coverage. Studies in The Netherlands and Finland have investigated polymorphism in the genes coding for two important virulence factors of
Bordetella pertussis
, pertactin and pertussis toxin, and identified the emergence and subsequent dominance in circulating strains of pertactin and toxin variants not found in the whole-cell vaccine (WCV). The study described here investigated whether such variation had occurred in the United Kingdom, which presently has low levels of pertussis. Sequence analysis of the genes for pertactin (
prnA
) and the pertussis toxin S1 subunit (
ptxA
) among isolates of
B. pertussis
from 285 United Kingdom patients, from 1920 to 1999, revealed three
prnA
variants,
prnA
(1),
prnA
(2), and
prnA
(3), and two
ptxA
variants,
ptxA
(1) and
ptxA
(2), showing differences in nucleic acid sequence. The proportion of pertactin gene types not included in the United Kingdom WCV, i.e.,
prnA
(2) and
prnA
(3), has increased in recent years and was found in 21 of 86 (24%) strains from the 1980s and 56 of 105 (53%) strains from the 1990s. To date, the presence of these nonvaccine
prnA
types has not been associated with a resurgence of pertussis in the United Kingdom. The distribution of
prnA
and
ptxA
types in The Netherlands, Finland, and the United Kingdom in the 1990s is distinct. The most striking difference in the United Kingdom isolates is that all 105 of the most recent circulating strains (from 1998 to 1999) are of a pertussis toxin type found in the United Kingdom WCV, i.e.,
ptxA
(1).
DuringTrypanosoma cruziInfection CD1d-Restricted NK T Cells Limit Parasitemia and Augment the Antibody Response to a Glycophosphoinositol-Modified Surface Protein
Authors: Duthie Malcolm S. , Wleklinski-Lee Monika , Smith Sherilyn , Nakayama Toshinori , Taniguchi Masaru , Kahn Stuart J.
Published: July 2002

Trypanosoma cruziis a protozoan parasite that chronically infects many mammalian species and in humans causes Chagas’ disease, a chronic inflammatory disease. The parasite expresses glycophosphoinositol (GPI), which potently stimulates interleukin 12 (IL-12) production. DuringT. cruziinfection IL-12, and possibly GPI, might stimulate NK T cells to affect the protective and chronic inflammatory responses. Here we report that duringT. cruziinfection CD1d-restricted NK T cells are stimulated as NK T-cell-deficient mice have greater parasitemia. Furthermore, duringT. cruziinfection the percentages of NK T cells in the liver and spleen become decreased for prolonged periods of time, and in vitro stimulation of NK T cells derived from livers of chronically infected mice, compared to uninfected mice, results in increased gamma interferon and IL-4 secretion. Moreover, in NK T-cell-deficient mice the chronic-phase antibody response to a GPI-modified surface protein is decreased. These results indicate that, during the acute infection, NK T cells limit parasitemia and that, during the chronic phase, NK T cells augment the antibody response. Thus, duringT. cruziinfection the quality of an individual’s NK T-cell response can affect the level of parasitemia and parasite tissue burden, the intensity of the chronic inflammatory responses, and possibly the outcome of Chagas’ disease.
PersistentEhrlichia chaffeensisInfection Occurs in the Absence of Functional Major Histocompatibility Complex Class II Genes
Authors: Ganta Roman Reddy , Wilkerson Melinda J. , Cheng Chuanmin , Rokey Aaron M. , Chapes Stephen K.
Published: July 2002

Human monocytic ehrlichiosis is an emerging tick-borne disease caused by the rickettsiaEhrlichia chaffeensis. We investigated the impact of two genes that control macrophage and T-cell function on murine resistance toE. chaffeensis. Congenic pairs of wild-type and toll-like receptor 4 (tlr4)- or major histocompatibility complex class II (MHC-II)-deficient mice were used for these studies. Wild-type mice cleared the infection within 2 weeks, and the response included macrophage activation and the synthesis ofE. chaffeensis-specific Th1-type immunoglobulin G response. The absence of a functionaltlr4gene depressed nitric oxide and interleukin 6 secretion by macrophages and resulted in short-term persistent infections for ≥30 days. In the absence of MHC-II alleles,E. chaffeensisinfections persisted throughout the entire 3-month evaluation period. Together, these data suggest that macrophage activation and cell-mediated immunity, orchestrated by CD4+T cells, are critical for conferring resistance toE. chaffeensis.
ohr, Encoding an Organic Hydroperoxide Reductase, Is an In Vivo-Induced Gene inActinobacillus pleuropneumoniae
Authors: Shea Robin J. , Mulks Martha H.
Published: July 2002

Actinobacillus pleuropneumoniaeis the causative agent of porcine pleuropneumonia, a disease characterized by pulmonary necrosis and hemorrhage caused in part by neutrophil degranulation. In an effort to understand the pathogenesis of this disease, we have developed an in vivo expression technology (IVET) system to identify genes that are specifically up-regulated during infection. One of the genes that we have identified as being induced in vivo isohr, encoding organic hydroperoxide reductase, an enzyme that could play a role in detoxification of organic hydroperoxides generated during infection. Among the 12 serotypes ofA.pleuropneumoniae,ohrwas found in only serotypes 1, 9, and 11. This distribution correlated with increased resistance to cumene hydroperoxide, an organic hydroperoxide, but not to hydrogen peroxide or to paraquat, a superoxide generator. Functional assays of Ohr activity demonstrated thatA.pleuropneumoniaeserotype 1 cultures, but not serotype 5 cultures, were able to degrade cumene hydroperoxide. InA.pleuropneumoniaeserotype 1, expression ofohrwas induced by cumene hydroperoxide, but not by either hydrogen peroxide or paraquat. In contrast, anohrgene from serotype 1 cloned intoA.pleuropneumoniaeserotype 5 was not induced by cumene hydroperoxide or by other forms of oxidative stress, suggesting the presence of a serotype-specific positive regulator ofohrinA.pleuropneumoniaeserotype 1.
Protective Immune Responses to the 42-Kilodalton (kDa) Region ofPlasmodium yoeliiMerozoite Surface Protein 1 Are Induced by the C-Terminal 19-kDa Region but Not by the Adjacent 33-kDa Region
Authors: Ahlborg Niklas , Ling Irene T. , Howard Wendy , Holder Anthony A. , Riley Eleanor M.
Published: July 2002

Vaccination of mice with the 42-kDa region ofPlasmodium yoeliimerozoite surface protein 1 (MSP142) or its 19-kDa C-terminal processing product (MSP119) can elicit protective antibody responses in mice. To investigate if the 33-kDa N-terminal fragment (MSP133) of MSP142also induces protection, the gene segment encoding MSP133was expressed as a glutathioneS-transferase (GST) fusion protein. C57BL/6 and BALB/c mice were immunized with GST-MSP133and subsequently challenged with the lethalP. yoeliiYM blood stage parasite. GST-MSP133failed to induce protection, and all mice developed patent parasitemia at a level similar to that in naive or control (GST-immunized) mice; mice immunized with GST-MSP119were protected, as has been shown previously. Specific prechallenge immunoglobulin G (IgG) antibody responses to MSP1 were analyzed by enzyme-linked immunosorbent assay and immunofluorescence. Despite being unprotected, several mice immunized with MSP133had antibody titers (of all IgG subclasses) that were comparable to or higher than those in mice that were protected following immunization with MSP119. The finding thatP. yoeliiMSP133elicits strong but nonprotective antibody responses may have implications for the design of vaccines for humans based onPlasmodium falciparumorPlasmodium vivaxMSP142.