bluuebunny/crossref_metadata_2025 huggingface dataset:
Potential Involvement of Gelatinases and Their Inhibitors inMannheimia haemolyticaPneumonia in Cattle
Authors: Starr Amanda E. , Dan Tonima , Minhas Kanwal , Shewen Patricia E. , Coomber Brenda L.
Published: July 2004

Mannheimia haemolyticainfection of the lower respiratory tract of cattle results in a bronchofibrinous pneumonia characterized by massive cellular influx and lung tissue remodeling and scarring. Since altered levels of gelatinases and their inhibitors have been detected in a variety of inflammatory conditions and are associated with tissue remodeling, we examined the presence of gelatinases in lesional and nonlesional lung tissue obtained from calves experimentally infected withM. haemolytica. Lesional tissue had elevated levels of progelatinase A and B and active gelatinase A and B when compared with nonlesional tissue obtained from the same lung lobe. In vitro,M. haemolyticaproducts stimulated production of gelatinase B, but not its activation, by bovine monocytes. Alveolar macrophages showed constitutive production of gelatinase B but no change in response toM. haemolyticaproducts. Bovine neutrophils exposed toM. haemolyticaproducts also released gelatinase B, and there was a significant increase in the activated form of this enzyme. These effects were virtually identical when recombinantO-sialoglycoprotease was used to stimulate these cells.M. haemolyticaproducts also enhanced the expression by bovine monocytes and alveolar macrophages of the tissue inhibitor of metalloproteinase 1. Our results provide evidence that matrix metalloproteinases are activated in lung lesions from cattle with shipping fever and thatM. haemolyticavirulence products induce production, release, and especially activation of gelatinase B by bovine inflammatory cells in vitro.
Induction of Maturation and Cytokine Release of Human Dendritic Cells byHelicobacter pylori
Authors: Kranzer Katharina , Eckhardt Alexander , Aigner Michael , Knoll Gertrud , Deml Ludwig , Speth Cornelia , Lehn Norbert , Rehli Michael , Schneider-Brachert Wulf
Published: July 2004

Helicobacter pyloricauses a persistent infection in the human stomach, which can result in chronic gastritis and peptic ulcer disease. Despite an intensive proinflammatory response, the immune system is not able to clear the organism. However, the immune escape mechanisms of this common bacterium are not well understood. We investigated the interaction betweenH. pyloriand human dendritic cells. Dendritic cells (DCs) are potent antigen-presenting cells and important mediators between the innate and acquired immune system. Stimulation of DCs with different concentrations ofH. pylorifor 8, 24, 48, and 72 h resulted in dose-dependent interleukin-6 (IL-6), IL-8, IL-10 and IL-12 production. Lipopolysaccharide (LPS) fromEscherichia coli, a known DC maturation agent, was used as a positive control. The cytokine release after stimulation with LPS was comparable to that induced byH. pyloriexcept for IL-12. After LPS stimulation IL-12 was only moderately released compared to the large amounts of IL-12 induced byH. pylori. We further investigated the potential ofH. pylorito induce maturation of DCs. Fluorescence-activated cell sorting analysis of cell surface expression of maturation marker molecules such as CD80, CD83, CD86, and HLA-DR revealed equal upregulation after stimulation withH. pylorior LPS. We found no significant differences betweenH. pyloriseropositive and seronegative donors of DCs with regard to cytokine release and upregulation of surface molecules. These data clearly demonstrate thatH. pyloriinduces a strong activation and maturation of human immature DCs.
CD4+Th1 Cells Induced by Dendritic Cell-Based Immunotherapy in Mice Chronically Infected withLeishmania amazonensisDo Not Promote Healing
Authors: Vanloubbeeck Yannick F. , Ramer Amanda E. , Jie Fei , Jones Douglas E.
Published: July 2004

The susceptibility of mice toLeishmania amazonensisinfection is thought to result from an inability to develop a Th1 response. Our data show that the low levels of gamma interferon (IFN-γ) produced by the draining lymph node (DLN) cells of chronically infected mice could be enhanced in vitro and in vivo withL. amazonensisantigen-pulsed bone marrow-derived dendritic cells (BM-DC) and the Th1-promoting cytokine interleukin-12 (IL-12). Given intralesionally to chronically infected mice, this treatment induced the upregulation of mRNA levels for IFN-γ, the transcription factor T-box expressed in T cells, and IL-12 receptor β2 in CD4+T cells from the DLN and an increase in parasite-specific immunoglobulin G2a in the serum. However, this Th1 response was not associated with healing, and the antigen-specific enhancement of IFN-γ production remained impaired in the DLN. However, addition of IL-12 to the in vitro recall response was able to recover this defect, suggesting that antigen-presenting cell-derived IL-12 production may be limited in infected mice. This was supported by the fact thatL. amazonensisamastigotes limited the production of IL-12p40 from BM-DC in vitro. Altogether, our data indicate that the immune response of mice chronically infected withL. amazonensiscan be enhanced towards a Th1 phenotype but that the presence of Th1 CD4+T cells does not promote healing. This suggests that the phenotype of the CD4+T cells may not always be indicative of protection toL. amazonensisinfection. Furthermore, our data support growing evidence that antigen-presenting cell function, such as IL-12 production, may limit the immune response inL. amazonensis-infected mice.
Production of the Subdomains of the
Plasmodium falciparum
Apical Membrane Antigen 1 Ectodomain and Analysis of the Immune Response
Authors: Lalitha P. V. , Ware Lisa A. , Barbosa Arnoldo , Dutta Sheetij , Moch J. Kathleen , Haynes J. David , Fileta Bader B. , White Charles E. , Lanar David E.
Published: July 2004

The apical membrane antigen 1 of
Plasmodium falciparum
is one of the leading candidate antigens being developed as a vaccine to prevent malaria. This merozoite transmembrane protein has an ectodomain that can be divided into three subdomains (D I, D II, and D III). We have previously expressed a major portion of this ectodomain and have shown that it can induce antibodies that prevent merozoite invasion into red blood cells in an in vitro growth and invasion assay. To analyze the antibody responses directed against the individual subdomains, we constructed six different genes that express each of the domains separately (D I, D II, or D III) or in combination with another domain (D I+II, D II+III, or D I+III). These proteins were purified and used to immunize rabbits to raise construct-specific antibodies. We demonstrated that D I+II induced a significant amount of the growth-inhibitory antibodies active in the growth and invasion assay.
A Specific Genomic Location within the
icm
/
dot
Pathogenesis Region of Different
Legionella
Species Encodes Functionally Similar but Nonhomologous Virulence Proteins
Authors: Feldman Michal , Segal Gil
Published: July 2004

Legionella pneumophila
, the major causative agent of Legionnaires' disease, is a facultative intracellular pathogen that grows within human macrophages and amoebae. Intracellular growth involves the formation of a replicative phagosome that requires the Icm/Dot type IV secretion system. Part of the
icm
/
dot
region in
L. pneumophila
contains the
icmTSRQPO
genes. The proteins encoded by the
icmR
and
icmQ
genes were shown to exhibit a chaperone-substrate relationship. Analysis of this region from other pathogenic
Legionella
species, i.e.,
L. micdadei
and
L. longbeachae
, indicated that the overall organization of this region is highly conserved, but it was found to contain a favorable site for gene variation. In the place where the
icmR
gene was expected to be located, other open reading frames that are nonhomologous to each other or to any entry in the GenBank database were found (
migAB
in
L. micdadei
and
ligB
in
L. longbeachae
). Examination of these unique genes revealed an outstanding phenomenon; by use of interspecies complementation, the
icmR
,
migB
, and
ligB
gene products were found to be functionally similar. In addition, the function of these proteins was usually dependent on the presence of the corresponding IcmQ proteins. Moreover, each of these proteins (IcmR, LigB, and MigB) was found to interact with the corresponding IcmQ proteins, and the genes encoding these proteins were found to be regulated by CpxR. This study reveals new evidence of gene variation occurring in the same genomic location within the
icm
/
dot
locus in various
Legionella
species. The genes found at this site were shown to be similarly regulated and to encode species-specific, nonhomologous, but functionally similar proteins.
Expression of the LspA1 and LspA2 Proteins by
Haemophilus ducreyi
Is Required for Virulence in Human Volunteers
Authors: Janowicz Diane M. , Fortney Kate R. , Katz Barry P. , Latimer Jo L. , Deng Kaiping , Hansen Eric J. , Spinola Stanley M.
Published: July 2004

Haemophilus ducreyi
colocalizes with polymorphonuclear leukocytes and macrophages and evades phagocytosis during experimental infection of human volunteers.
H. ducreyi
contains two genes,
lspA1
and
lspA2
, which encode predicted proteins of 456 and 543 kDa, respectively. Compared to its wild-type parent, an
lspA1 lspA2
double mutant does not inhibit phagocytosis by macrophage and myelocytic cell lines in vitro and is attenuated in an experimental rabbit model of chancroid. To test whether expression of LspA1 and LspA2 was necessary for virulence in humans, six volunteers were experimentally infected. Each volunteer was inoculated with three doses (ranging from 85 to 112 CFU) of the parent (35000HP) in one arm and three doses (ranging from 60 to 822 CFU) of the mutant (35000HPΩ12) in the other arm. The papule formation rates were 88% (95% confidence interval [95% CI], 76.8 to 99.9%) at 18 parent sites and 72% (95% CI, 44.4 to 99.9%) at 18 mutant sites (
P
= 0.19). However, papules were significantly smaller at mutant sites (mean size, 24.8 mm
2
) than at parent sites (mean size, 39.1 mm
2
) 24 h after inoculation (
P
= 0.0002). The pustule formation rates were 44% (95% CI, 5.8 to 77.6%) at parent sites and 0% (95% CI, 0 to 39.4%) at mutant sites (
P
= 0.009). With the caveat that biosafety regulations preclude testing of a complemented mutant in human subjects, these results indicate that expression of LspA1 and LspA2 facilitates the ability of
H. ducreyi
to initiate disease and to progress to pustule formation in humans.
Protection against Pneumococcal Pneumonia in Mice by Monoclonal Antibodies to Pneumolysin
Authors: García-Suárez María del Mar , Cima-Cabal María Dolores , Flórez Noelia , García Pilar , Cernuda-Cernuda Rafael , Astudillo Aurora , Vázquez Fernando , De Los Toyos Juan R. , Méndez F. Javier
Published: July 2004

Pneumolysin (PLY) is an important virulence factor of
Streptococcus pneumoniae
. We examined the ability of three murine monoclonal antibodies (MAbs) to PLY (PLY-4, PLY-5, and PLY-7) to affect the course of pneumococcal pneumonia in mice. The intravenous administration of antibodies PLY-4 and PLY-7 protected the mice from the lethal effect of the purified toxin. Mice treated with PLY-4 before intranasal inoculation of
S. pneumoniae
type 2 survived longer (median survival time, 100 h) than did untreated animals (median survival time, 60 h) (
P
< 0.0001). The median survival time for mice treated with a combination of PLY-4 and PLY-7 was 130 h, significantly longer than that for mice given isotype-matched indifferent MAbs (
P
= 0.0288) or nontreated mice (
P
= 0.0002). The median survival time for mice treated with a combination of three MAbs was significantly longer (>480 h) than that for mice treated with PLY-5 (48 h;
P
< 0.0001), PLY-7 (78 h;
P
= 0.0007), or PLY-4 (100 h;
P
= 0.0443) alone. Similarly, the survival rate for mice treated with three MAbs (10 of 20 mice) was significantly higher than the survival rate obtained with PLY-5 (1 of 20;
P
= 0.0033), PLY-4 (2 of 20;
P
= 0.0138), or PLY-7 (3 of 20;
P
= 0.0407) alone. These results suggest that anti-PLY MAbs act with a synergistic effect. Furthermore, MAb administration was associated with a significant decrease in bacterial lung colonization and lower frequencies of bacteremia and tissue injury with respect to the results for the control groups.
Icm/Dot-Independent Entry of
Legionella pneumophila
into Amoeba and Macrophage Hosts
Authors: Bandyopadhyay Purnima , Xiao Huifang , Coleman Hope A. , Price-Whelan Alexa , Steinman Howard M.
Published: July 2004

Legionella pneumophila
, the causative agent of Legionnaires' disease, expresses a type IVB secretion apparatus that translocates bacterial proteins into amoeba and macrophage hosts. When stationary-phase cultures are used to infect hosts, the type IVB apparatus encoded by the
icm
/
dot
genes is required for entry, delay of phagosome-lysosome fusion, and intracellular multiplication within host cells. Null mutants with mutations in
icm
/
dot
genes are defective in these phenotypes. Here a new model is described in which hosts are infected with stationary-phase cultures that have been incubated overnight in pH 6.5 buffer. This model is called Ers treatment because it enhances the resistance to acid, hydrogen peroxide, and antibiotic stress beyond that of stationary-phase cultures. Following Ers treatment entry into amoeba and macrophage hosts does not require
dotA
, which is essential for
Legionella
virulence phenotypes when hosts are infected with stationary-phase cultures,
dotB
,
icmF
,
icmV
, or
icmX
. Defective host entry is also suppressed for null mutants with mutations in the KatA and KatB catalase-peroxidase enzymes, which are required for proper intracellular growth in amoeba and macrophage hosts. Ers treatment-induced suppression of defective entry is not associated with increased bacterial adhesion to host cells or with morphological changes in the bacterial envelope but is dependent on protein expression during Ers treatment. By using proteomic analysis, Ers treatment was shown to induce a protein predicted to contain eight tetratricopeptide repeats, a motif previously implicated in enhanced entry of
L. pneumophila
. Characterization of Ers treatment-dependent changes in expression is proposed as an avenue for identifying
icm
/
dot
-independent factors that function in the entry of
Legionella
into amoeba and macrophage hosts.
A STAT4-Dependent Th1 Response Is Required for Resistance to the Helminth ParasiteTaenia crassiceps
Authors: Rodríguez-Sosa Miriam , Saavedra Rafael , Tenorio Eda P. , Rosas Lucia E. , Satoskar Abhay R. , Terrazas Luis I.
Published: July 2004

To determine the role of STAT4-dependent Th1 responses in the regulation of immunity to the helminth parasiteTaenia crassiceps, we monitored infections with this parasite in resistant mice lacking the STAT4 gene. WhileT. crassiceps-infected STAT4+/+mice rapidly resolved the infection, STAT4−/−mice were highly susceptible to infection and displayed large parasite loads. Moreover, the inability of STAT4−/−mice to control the infection was associated with the induction of an antigen-specific Th2-type response characterized by significantly higher levels of Th2-associated immunoglobulin G1 (IgG1) and total IgE as well as interleukin-4 (IL-4), IL-10, and IL-13 than those in STAT4+/+mice, who produced significantly more gamma interferon. Furthermore, early after infection, macrophages from STAT4−/−mice produced lower levels of the pro-inflammatory cytokines IL-12, tumor necrosis factor alpha, IL-1β, and nitric oxide (NO) than those from STAT4+/+mice, suggesting a pivotal role for macrophages in mediating protection against cysticercosis. These findings demonstrate a critical role for the STAT4 signaling pathway in the development of a Th1-type immune response that is essential for mediating protection against the larval stage ofT. crassicepsinfection.
Toll-Like Receptor 2 Represses Nonpilus Adhesin-Induced Signaling in Acute Infections with the
Pseudomonas aeruginosa pilA
Mutant
Authors: Lorenz Eva , Chemotti Diana C. , Vandal Karen , Tessier Philippe A.
Published: July 2004

Expression of pili and associated proteins is an important means of host invasion by bacterial pathogens. Recent evidence has suggested that the binding of
Pseudomonas aeruginosa
through nonpilus adhesins may also be important in respiratory diseases, since adhesins bind mucins. Using wild-type C57BL/6 and TLR2KO mice, we compared the induction levels of the host response to
P. aeruginosa
that either expressed pili or lacked pilus expression due to a mutation in the structural gene
pilA
. In C57BL/6 mice, deletion of pili led to a decreased immune response, evidenced by a lower secretion of cytokines and a lack of neutrophil chemotaxis. By contrast, the
P. aeruginosa pilA
mutant induced a hyperresponsive phenotype in TLR2KO mice. TLR2KO mice showed an increased number of neutrophils in lavage fluid compared to the levels seen when either mouse strain was exposed to wild-type
P. aeruginosa
. Further analysis indicated that the increased neutrophil influx was associated with an increased expression of calgranulins, possibly through an induction of Toll-like receptor 4 (TLR4) expression. The hyperresponsive phenotype of TLR2KO mice exposed to the
P. aeruginosa pilA
mutant was associated with TLR4 induction and indicated that nonpilus adhesin-induced signaling was repressed by TLR2 function and, if not blocked by the host, could induce airway hyperresponsiveness.
UropathogenicEscherichia coliTriggers Oxygen-Dependent Apoptosis in Human Neutrophils through the Cooperative Effect of Type 1 Fimbriae and Lipopolysaccharide
Authors: Blomgran Robert , Zheng Limin , Stendahl Olle
Published: July 2004

Type 1 fimbriae are the most commonly expressed virulence factor on uropathogenicEscherichia coli. In addition to promoting avid bacterial adherence to the uroepithelium and enabling colonization, type 1 fimbriae recruit neutrophils to the urinary tract as an early inflammatory response. Using clinical isolates of type 1 fimbriatedE. coliand an isogenic type 1 fimbria-negative mutant (CN1016) lacking the FimH adhesin, we investigated if these strains could modulate apoptosis in human neutrophils. We found thatE. coliexpressing type 1 fimbriae interacted with neutrophils in a mannose- and lipopolysaccharide (LPS)-dependent manner, leading to apoptosis which was triggered by the intracellular generation of reactive oxygen species. This induced neutrophil apoptosis was abolished by blocking FimH-mediated attachment, by inhibiting NADPH oxidase activation, or by neutralizing LPS. In contrast, CN1016, which did not adhere to or activate the respiratory burst of neutrophils, delayed the spontaneous apoptosis in an LPS-dependent manner. This delayed apoptosis could be mimicked by adding purified LPS and was also observed by using fimbriated bacteria in the presence ofd-mannose. These results suggest that LPS is required forE. colito exert both pro- and antiapoptotic effects on neutrophils and that the difference in LPS presentation (i.e., with or without fimbriae) determines the outcome. The present study showed that there is a fine-tuned balance between type 1 fimbria-induced and LPS-mediated delay of apoptosis in human neutrophils, in which altered fimbrial expression on uropathogenicE. colidetermines the neutrophil survival and the subsequent inflammation during urinary tract infections.
phgABC
, a Three-Gene Operon Required for Growth of
Streptococcus pneumoniae
in Hyperosmotic Medium and In Vivo
Authors: Brown Jeremy S. , Gilliland Sarah M. , Basavanna Shilpa , Holden David W.
Published: July 2004

To cause disease, bacterial pathogens need to be able to adapt to the physiological conditions found within the host, including an osmolality of approximately 290 mosmol kg
−1
. While investigating
Streptococcus pneumoniae
genes contained within pneumococcal pathogenicity island 1, we identified a three-gene operon of unknown function termed
phgABC
. PhgC has a domain with similarity to diacylglycerol kinases of eukaryotes and is the first described member of a family of related proteins found in many gram-positive bacteria.
phgA
and
phgC
mutant strains were constructed by insertional duplication mutagenesis and found to have impaired growth under conditions of high osmotic and oxidative stress. The compatible solutes proline and glycine betaine improved growth of the wild-type and the
phgA
mutant strains in hyperosmolar medium, and when analyzed by electron microscopy, the cellular morphology of the
phgA
mutant strain was unaffected by osmotic stress. The
phgA
and
phgC
mutant strains were reduced in virulence in models of both systemic and pulmonary infection. As the virulence of the
phgA
mutant strain was not restored in

gp91phox
−/−

mice and the
phgA
and
phgC
mutant strains had reduced growth in both blood and serum, the reduced virulence of these strains is unlikely to be due to increased sensitivity to the respiratory burst of phagocytes but is, instead, due to impaired growth at physiological osmolality.
Pneumocystis carinii
Cell Wall Biosynthesis Kinase Gene
CBK1
Is an Environmentally Responsive Gene That Complements Cell Wall Defects of
cbk
-Deficient Yeast
Authors: Kottom Theodore J. , Limper Andrew H.
Published: July 2004

Pneumocystis
species remain an important cause of life-threatening pneumonia in immunocompromised hosts, including those with AIDS. Responses of the organism to environmental cues both within the lung and elsewhere have been poorly defined. Herein, we report the identification of a cell wall biosynthesis kinase gene (
CBK1
) homologue in
Pneumocystis carinii
, isolated by differential display PCR, that is expressed optimally at physiological pH (7 to 8) as opposed to more acidic environments. Expression of
Pneumocystis CBK1
was also induced by contact with lung epithelial cells and extracellular matrix. Translation of this gene revealed extensive homology to other fungal CBK1 kinases.
Pneumocystis CBK1
expression was equal in the cyst and trophic life forms of the organisms. We further demonstrate that
Pneumocystis CBK1
expressed in
cbk1Δ Saccharomyces cerevisiae
cells restored defective cell wall separation during proliferation. Consistent with this,
Pneumocystis CBK1
expression also stimulated transcription of the
CTS1
chitinase in
cbk1Δ
mutant yeast cells, an event necessary for cell wall separation. In addition,
Pneumocystis CBK1
cDNA supported normal mating projection formation in response to α-factor in the
cbk1Δ
yeast cells. Site-directed mutations of serine-303 and threonine-494, potential regulatory phosphorylation sites in
Pneumocystis
CBK1, abolished mating projection formation, indicating a role for these amino acid residues in CBK1 activity. These findings indicate that
Pneumocystis CBK1
is an environmentally responsive gene that may function in signaling pathways necessary for cell growth and mating.
Role of the Two-Component Regulator CpxAR in the Virulence of
Salmonella enterica
Serotype Typhimurium
Authors: Humphreys Sue , Rowley Gary , Stevenson Andrew , Anjum Muna F. , Woodward Martin J. , Gilbert Stephen , Kormanec Jan , Roberts Mark
Published: July 2004

The CpxAR (Cpx) two-component regulator controls the expression of genes in response to a variety of environmental cues. The Cpx regulator has been implicated in the virulence of several gram-negative pathogens, although a role for Cpx in vivo has not been demonstrated directly. Here we investigate whether positive or negative control of gene expression by Cpx is important for the pathogenesis of
Salmonella enterica
serotype Typhimurium. The Cpx signal pathway in serotype Typhimurium was disrupted by insertional inactivation of the
cpxA
and
cpxR
genes. We also constitutively activated the Cpx pathway by making an internal in-frame deletion in
cpxA
(a
cpxA*
mutation). Activation of the Cpx pathway inhibited induction of the envelope stress response pathway controlled by the alternative sigma factor σ
E
(encoded by
rpoE
). Conversely, the Cpx pathway was highly up-regulated (>40-fold) in a serotype Typhimurium
rpoE
mutant. The
cpxA
* mutation, but not the
cpxA
or the
cpxR
mutation, significantly reduced the capacity of serotype Typhimurium to adhere to and invade eucaryotic cells, although intracellular replication was not affected. The
cpxA
and
cpxA*
mutations significantly impaired the ability of serotype Typhimurium to grow in vivo in mice. To our knowledge, this is the first demonstration that the Cpx system is important for a bacterial pathogen in vivo.
ChronicHelicobacter pyloriInfection with Sydney Strain 1 and a Newly Identified Mouse-Adapted Strain (Sydney Strain 2000) in C57BL/6 and BALB/c Mice
Authors: Thompson Lucinda J. , Danon Stephen J. , Wilson John E. , O'Rourke Jani L. , Salama Nina R. , Falkow Stanley , Mitchell Hazel , Lee Adrian
Published: July 2004

The mouse model ofHelicobacter pylori-induced disease using Sydney strain 1 (SS1) has been used extensively inHelicobacterresearch. Herein we describe the isolation and characterization of a new mouse-colonizing strain for use in comparative studies. One strain capable of persistent mouse colonization was isolated from a total of 110 clinical isolates and is named here SS2000 (Sydney strain 2000). Genome typing revealed a number of differences between SS1 and SS2000 as well as between them and the respective original clinical isolates. In particular, SS2000 lacked the entirecagpathogenicity island, while SS1 contained all 27 genes of the island. C57BL/6 and BALB/c mice were infected with SS1 or SS2000 or were treated with broth medium (controls). After 6 months host-specific effects were evident, including lower colonization levels in the BALB/c animals. Few pathological differences were observed between SS1- and SS2000-infected animals. However, by 15 months postinfection, SS1-infected C57BL/6 mice had developed more severe gastritis than the SS2000-infected animals. In contrast SS2000-infected BALB/c mice showed increased accumulation of mucosa-associated lymphoid tissue compared to those infected with SS1. This improved comparative model ofH. pylori-induced disease allowed dissection of both host and strain effects and thus will prove useful in further studies.
Characterization of theStreptococcus mutansP1 Epitope Recognized by Immunomodulatory Monoclonal Antibody 6-11A
Authors: Rhodin Nikki R. , Cutalo Jenny M. , Tomer Kenneth B. , McArthur William P. , Brady L. Jeannine
Published: July 2004

Monoclonal antibody (MAb) 6-11A directed againstStreptococcus mutanssurface adhesin P1 was shown previously to influence the mucosal immunogenicity of this organism in BALB/c mice. The specificity of anti-P1 serum immunoglobulin G (IgG) and secretory IgA antibodies and the subclass distribution of anti-P1 serum IgG antibodies were altered, and the ability of elicited serum antibodies to inhibitS. mutansadherence in vitro was in certain cases increased. MAb 6-11A is known to recognize an epitope dependent on the presence of the proline-rich region of the protein, although it does not bind directly to the isolated P-region domain. In this report, we show that MAb 6-11A recognizes a complex discontinuous epitope that requires the simultaneous presence of the alanine-rich repeat domain (A-region) and the P-region. Formation of the core epitope requires the interaction of these segments of P1. Residues amino terminal to the A-region also contributed to recognition by MAb 6-11A but were not essential for binding. Characterization of the MAb 6-11A epitope will enable insight into potential mechanisms of immunomodulation and broaden our understanding of the tertiary structure of P1.
Contribution of the Alanine-Rich Region of
Streptococcus mutans
P1 to Antigenicity, Surface Expression, and Interaction with the Proline-Rich Repeat Domain
Authors: Seifert Trevor B. , Bleiweis Arnold S. , Brady L. Jeannine
Published: July 2004

Streptococcus mutans
is considered to be the major etiologic agent of human dental caries. Attachment of
S. mutans
to the tooth surface is required for the development of caries and is mediated, in part, by the 185-kDa surface protein variously known as antigen I/II, PAc, and P1. Such proteins are expressed by nearly all species of oral streptococci. Characteristics of P1 include an alanine-rich repeat region and a centrally located proline-rich repeat region. The proline-rich region of P1 has been shown to be important for the translational stability and translocation of P1 through the bacterial membrane. We show here that (i) several anti-P1 monoclonal antibodies require the simultaneous presence of the alanine-rich and proline-rich regions for binding, (ii) the proline-rich region of P1 interacts with the alanine-rich region, (iii) like the proline-rich region, the alanine-rich region is required for the stability and translocation of P1, (iv) both the proline-rich and alanine-rich regions are required for secretion of P1 in
Escherichia coli
, and (v) in
E. coli
, P1 is secreted in the absence of SecB.
Novel Phage Display-Based Subtractive Screening To Identify Vaccine Candidates ofBrugia malayi
Authors: Gnanasekar Munirathinam , Rao Kakaturu V. N. , He Yi-Xun , Mishra Pankaj K. , Nutman Thomas B. , Kaliraj Perumal , Ramaswamy Kalyanasundaram
Published: July 2004

This study describes a novel phage display method based on an iterative subtraction strategy to identify candidate vaccine antigens ofBrugia malayi. A cDNA library of the infective larval stage ofB. malayiexpressed on the surface of T7 phage was sequentially screened with sera samples from human subjects showing different manifestations of the disease. Antigens that selectively and specifically bind to immune sera were then enriched using a multi-step panning procedure. This strategy identified five antigens, four of which were previously reported (ALT-2, TPX-2, VAH and COX-2) and the other one was a novel cuticular collagen (Col-4). Sera from immune individuals specifically recognized all the five antigens. However, ALT-2 appeared to be the most predominantly recognized antigen by the immune sera. Therefore, it was decided to evaluate the vaccine potential of recombinant ALT-2 (rALT-2) in a mouse and jird model. The results presented show that immunization with rALT-2 conferred over 73% protection against a challenge infection in the jird model and over 64% protection in the mouse model. The present study suggests that phage display-based cDNA screening may be a powerful tool to identify candidate vaccine antigens of infectious agents.
Adherence to, Invasion by, and Cytokine Production in Response to Serotype VIII Group B Streptococci
Authors: Mikamo Hiroshige , Johri Atul K. , Paoletti Lawrence C. , Madoff Lawrence C. , Onderdonk Andrew B.
Published: July 2004

The adherence to and invasion of the human epithelial cell line A549 by group B streptococcus (GBS) serotype VIII strains were compared with those of serotype III strains by a conventional method and the dynamic in vitro attachment and invasion system. Twenty GBS strains, including nine vaginal isolates and one invasive isolate each of serotypes III and VIII, were used in the conventional attachment and invasion assay. Adherence to and invasion of A549 cells by serotype VIII GBS strains were significantly greater (P< 0.0001) than those by serotype III strains for both the invasive strain and vaginal isolates. Cytokine production by A549 cells following stimulation with GBS serotypes III and VIII or their purified capsular polysaccharides (CPS) was measured. Serotype III strains stimulated significantly greater tumor necrosis factor alpha (TNF-α) (P< 0.0001) and interleukin-10 (IL-10) (P< 0.05) production than did serotype VIII strains. IL-8 production in response to serotype VIII was significantly higher (P< 0.001) than that in response to serotype III. TNF-α, IL-8, and IL-10 production was greater in A549 cells infected with GBS than in the untreated control cells. TNF-α production was significantly greater (P< 0.005) after stimulation with purified GBS serotype III CPS than after stimulation with serotype VIII CPS, a result similar to that after stimulation with whole GBS. IL-12 production by A549 cells was observed only in response to infection with GBS serotype III, resulting in the possibility of a greater TH1 response in serotype III GBS. These results suggest differences in immune responses to infection with GBS serotypes III and VIII.
Experimental Infections of Neonatal Mice with Cysts of
Giardia lamblia
Clone GS/M-83-H7 Are Associated with an Antigenic Reset of the Parasite
Authors: von Allmen N. , Bienz M. , Hemphill A. , Müller N.
Published: July 2004

Transmission of the protozoan parasite
Giardia lamblia
from one to another host individuum occurs through peroral ingestion of cysts which, following excystation in the small intestine, release two trophozoites each. Many studies have focused on the major surface antigen, VSP (for variant surface protein), which is responsible for the antigenic variability of the parasite. By using trophozoites of
G. lamblia
clone GS/M-83-H7 (expressing VSP H7) and the neonatal mouse model for experimental infections, we quantitatively assessed the process of antigenic variation of the parasite on the transcriptional level. In the present study, variant-specific regions identified on different GS/M-83-H7
vsp
sequences served as targets for quantitative reverse transcription-PCR to monitor alterations in
vsp
mRNA levels during infection. Respective results demonstrated that antigenic switching of both the duodenal trophozoite and the cecal cyst populations was associated with a massive reduction in
vsp
H7 mRNA levels but not with a simultaneous increase in transcripts of any of the subvariant
vsp
genes analyzed. Most importantly, we also explored giardial variant-type formation and
vsp
mRNA levels after infection of mice with cysts. This infection mode led to an antigenic reset of the parasite in that a VSP H7-negative inoculum “converted” into a population of intestinal trophozoites that essentially consisted of the original VSP H7 type. This antigenic reset appears to be associated with excystation rather than with a selective process which favors expansion of a residual population of VSP H7 types within the antigenically diversified cyst inoculum. Based on these findings, the VSP H7 type has to be regarded as a predominant variant of
G. lamblia
clone GS/M-83-H7 which (re-)emerges during early-stage infection and may contribute to an optimal establishment of the parasite within the intestine of the experimental murine host.
Identification and Characterization of a
Neospora caninum
Microneme-Associated Protein (NcMIC4) That Exhibits Unique Lactose-Binding Properties
Authors: Keller Nadine , Riesen Michèle , Naguleswaran Arunasalam , Vonlaufen Nathalie , Stettler Rebecca , Leepin Angela , Wastling Jonathan M. , Hemphill Andrew
Published: July 2004

Microneme proteins have been shown to play an important role in the early phase of host cell adhesion, by mediating the contact between the parasite and host cell surface receptors. In this study we have identified and characterized a lectin-like protein of
Neospora caninum
tachyzoites which was purified by α-lactose-agarose affinity chromatography. Upon separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this lactose-binding protein migrated at 70 and 55 kDa under reducing and nonreducing conditions, respectively. Immunofluorescence and immunogold electron microscopy with affinity-purified antibodies showed that the protein was associated with the tachyzoite micronemes. Mass spectrometry analyses and expressed sequence tag database mining revealed that this protein is a member of the
Neospora
microneme protein family; the protein was named NcMIC4 (
N. caninum
microneme protein 4). Upon two-dimensional gel electrophoresis, NcMIC4 separated into seven distinct isoforms. Incubation of extracellular parasites at 37°C resulted in the secretion of NcMIC4 into the medium as a soluble protein, and the secreted protein exhibited a slightly reduced
M
r
but retained its lactose-binding properties. Immunofluorescence was used to investigate the temporal and spatial distribution of NcMIC4 in tachyzoites entering their host cells and showed that reexpression of NcMIC4 took place 30 min after entry into the host cell. Incubation of secreted fractions and purified NcMIC4 with Vero cells demonstrated binding of NcMIC4 to Vero cells as well as binding to chondroitin sulfate A glycosaminoglycans.
Murine Model of Pulmonary Anthrax: Kinetics of Dissemination, Histopathology, and Mouse Strain Susceptibility
Authors: Lyons C. Rick , Lovchik Julie , Hutt Julie , Lipscomb Mary F. , Wang Eugenia , Heninger Sara , Berliba Lucy , Garrison Kristin
Published: July 2004

Bioweapons are most often designed for delivery to the lung, although this route is not the usual portal of entry for many of the pathogens in the natural environment. Vaccines and therapeutics that are efficacious for natural routes of infection may not be effective against the pulmonary route. Pulmonary models are needed to investigate the importance of specific bacterial genes in virulence, to identify components of the host immune system that are important in providing innate and acquired protection, and for testing diagnostic and therapeutic strategies. This report describes the characteristics of host andBacillus anthracisinteractions in a murine pulmonary-infection model. The infective dose varied depending on the route and method of inoculation. The germination process in the lung began within 1 h of inoculation into the lung, although growth within the lung was limited.B. anthraciswas found in the lung-associated lymph nodes ∼5 h after infection. Minimal pneumonitis was associated with the lung infection, but significant systemic pathology was noted after dissemination. Infected mice typically succumbed to infection ∼3 to 4 days after inoculation. The 50% lethal doses differed among inbred strains of mice, but within a given mouse strain, neither the age nor the sex of the mice influenced susceptibility toB. anthracis.
The
luxS
Gene Is Not Required for
Borrelia burgdorferi
Tick Colonization, Transmission to a Mammalian Host, or Induction of Disease
Authors: Blevins Jon S. , Revel Andrew T. , Caimano Melissa J. , Yang Xiaofeng F. , Richardson James A. , Hagman Kayla E. , Norgard Michael V.
Published: July 2004

luxS
mutants of
Borrelia burgdorferi
strain 297 naturally colonized their arthropod (
Ixodes scapularis
) vector, were maintained in ticks throughout the molting process (larvae to nymphs), were tick transmitted to uninfected mice, and elicited histopathology in mice indistinguishable from that induced by wild-type
B. burgdorferi
.
Characterization of Adenylate Cyclase-Hemolysin Gene Duplication in a
Bordetella pertussis
Isolate
Authors: Dalet Karine , Weber Christian , Guillemot Laurent , Njamkepo Elisabeth , Guiso Nicole
Published: July 2004

We describe a clinical isolate of
Bordetella pertussis
, the agent responsible for whooping cough, composed of at least two clones harboring one or two copies of the
cya
locus encoding one of the major toxins, adenylate cyclase-hemolysin. No difference was observed between the two clones in murine and cellular models, probably due to the high instability of the
cya
locus duplication.
Inactivation of the
ciaH
Gene in
Streptococcus mutans
Diminishes Mutacin Production and Competence Development, Alters Sucrose-Dependent Biofilm Formation, and Reduces Stress Tolerance
Authors: Qi Fengxia , Merritt Justin , Lux Renate , Shi Wenyuan
Published: July 2004

Many clinical isolates of
Streptococcus mutans
produce peptide antibiotics called mutacins. Mutacin production may play an important role in the ecology of
S. mutans
in dental plaque. In this study, inactivation of a histidine kinase gene,
ciaH
, abolished mutacin production. Surprisingly, the same mutation also diminished competence development, stress tolerance, and sucrose-dependent biofilm formation.
Chlamydia pneumoniae
Decreases Smooth Muscle Cell Proliferation through Induction of Prostaglandin E
2
Synthesis
Authors: Rödel Jürgen , Prochnau Dirk , Prager Katrin , Baumert Jürgen , Schmidt Karl-Hermann , Straube Eberhard
Published: July 2004

Chlamydia pneumoniae
may modulate the proliferation of smooth muscle cells (SMC) in atherosclerotic plaques. Conditioned medium from
C. pneumoniae-
infected SMC decreased the proliferation of uninfected SMC. Treatment of infected cells with the cyclooxygenase-2 inhibitor NS-398 {
N
-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide} suppressed the up-regulation of prostaglandin E
2
(PGE
2
) and abolished the antimitogenic effect of conditioned medium, suggesting that
C. pneumoniae
can decrease SMC proliferation via stimulation of PGE
2
synthesis.
Role of Flagellum and Motility in Pathogenesis of
Vibrio vulnificus
Authors: Lee Jong-Ho , Rho Jong Bok , Park Kyung-Je , Kim Chang Beom , Han Yang-Soo , Choi Sang Ho , Lee Kyu-Ho , Park Soon-Jung
Published: July 2004

To assess the role of the flagellum which was detected by immunoscreening of surface proteins of
Vibrio vulnificus
, an
flgE
-deleted mutant was constructed and tested for its pathogenicity. The ability of this nonmotile mutant to adhere to INT-407 cells and its role in biofilm were decreased, as was its lethality to mice.
Intact Purine Biosynthesis Pathways Are Required for Wild-Type Virulence of
Brucella abortus
2308 in the BALB/c Mouse Model
Authors: Alcantara Rosemarie B. , Read Richard D. A. , Valderas Michelle Wright , Brown Timothy D. , Roop R. Martin
Published: July 2004

Brucella abortus
2308 derivatives with mini-Tn
5
insertions in
purE
,
purL
, and
purD
display significant attenuation in the BALB/c mouse model, while isogenic mutants with mini-Tn
5
insertions in
pheA
,
trpB
, and
dagA
display little or no attenuation in cultured murine macrophages or mice. These experimental findings confirm the importance of the purine biosynthesis pathways for the survival and replication of the brucellae in host macrophages. In contrast to previous reports, however, these results indicate that exogenous tryptophan and phenylalanine are available for use by the brucellae in the phagosomal compartment.
Off seasonal and pre-seasonal assessment of circulating energy sources during prolonged running at the anaerobic threshold in competitive triathletes
Authors: Knoepfli B , Riddell M C , Ganzoni E , Burki A , Villiger B , von Duvillard S P
Published: July 2004

Objectives:To compare changes in circulating energy sources during prolonged exercise in off season (OS) and pre-season (PS) training of triathletes.

Methods:Nine athletes of the Swiss national triathlon team (three female, mean (SD) age 28.7 (4.9) years, height 169.8 (6.0) cm, weight 57.0 (6.2) kg, V˙o2max66.5 (5.3) ml/min/kg; six male, mean (SD) age 24.0 (4.1) years, height 181.4 (6.9) cm, weight 73.5 (6.0) kg, V˙o2max75.9 (4.9) ml/min/kg) were tested twice (2.5 months apart) during a 25 km aerobic capacity test run at the end of the OS and just before the season. The average training load during the OS was 9.9 h/week, and this increased to 14.4 h/week in the PS. With heart rates as reference, exercise intensity during the aerobic capacity test was 97.0 (4.9)% of the anaerobic threshold and 91.2 (4.5)% of V˙o2max. Blood samples were collected before, during, and after the aerobic capacity test. Samples were collected every 5 km during three minute rest intervals.

Results:Blood was analysed for triglyceride (TG), free fatty acids, cholesterol, high density lipoprotein cholesterol, glucose, insulin, lactate, and changes in plasma volume. A two factor (season by distance) repeated measures analysis of variance revealed an increase in capacity for prolonged exercise in the PS by a decrease in running intensity during the aerobic capacity test (% of speed at 2.0 mmol/l lactate threshold, p  =  0.008), an increase in running speed at the anaerobic threshold (p  =  0.003) and at 4.0 and 2.0 mmol/l (p<0.001) of the lactate threshold. A significant season by distance interaction was found for TG (p<0.001). TG concentrations peaked at 5 km and decreased logarithmically throughout the OS (1.48 (0.34) to 0.86 (0.20) mmol/l) and PS (1.90 (0.31) to 0.73 (0.18) mmol/l) tests. From the OS to the PS, there was an increase in the difference in TG at 5–15 km with a concomitant increase at 2.0 mmol/l of the lactate threshold. The peak TG concentrations at 5 km followed by a logarithmic decrease suggest that TG may also provide circulating energy. A greater logarithmic decrease in TG occurred in the PS than in the OS, indicating a higher rate of use. There was an increase in the difference in TG at 5–15 km similar to the increase in the speed at 2.0 mmol/l of the lactate threshold between the two seasons. Glucose, insulin, lactate, and free fatty acids were similar in the two seasons.

Conclusion:Free fatty acid and TG concentrations were much higher than expected, and the two training seasons showed significantly different patterns of TG concentration during prolonged running. These responses may be related to aerobic capacity of prolonged exercise.
Shoulder pathoanatomy in marathon kayakers
Authors: Hagemann G , Rijke A M , Mars M
Published: July 2004

Objectives:To determine the prevalence of soft and hard tissue abnormalities and their interrelations in the shoulders of marathon kayakers and to examine the pathoanatomical factors that predispose these athletes to injury.

Methods:Fifty two long distance kayakers completed a questionnaire. Their shoulders were examined for range of motion, pain, and stability using a standard set of 10 clinical tests. The shoulder was subsequently scanned by magnetic resonance imaging (MRI) in three planes and evaluated for evidence of injury or other abnormality. The relation of clinical symptoms and MRI findings was investigated with respect to kayaker’s age, number of years kayaking, and number of marathon races completed.

Results:Thirty subjects were asymptomatic at the time of scanning, and twenty two showed symptoms of pain and/or instability. MRI showed acromioclavicular hypertrophy, acromial or clavicular spur, supraspinatus tendinitis, and partial tear of the supraspinatus as the most common abnormalities. Kayaker’s age, number of years kayaking, and number of races completed did not relate significantly to symptoms or to the presence of an abnormality on MRI scan. Of all the pathoanatomical findings that are reported to predispose to rotator cuff injury, only acromial and clavicular spurs were found to correlate highly with supraspinatus muscle pathology.

Conclusions:Rotator cuff injuries make up a large portion of the injuries seen in marathon kayakers, about twice the number reported for sprint kayakers. These injuries are the result of secondary impingement factors associated with overuse, possibly specific to kayakers, and not the result of bony restrictions around the shoulder joint. Acromioclavicular hypertrophy is a common finding in marathon kayakers, but is possibly the result of portaging or a previous injury.