bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence of lactoferrin transportation into blood circulation from intestine via lymphatic pathway in adult rats
Authors: Takeuchi Takashi , Kitagawa Hiroshi , Harada Etsumori
Published: February 2004

Using adult rats, the characteristic transporting system for lactoferrin (LF) from intestinal lumen into the blood circulation was investigated. The rats were randomly divided into two groups, a non‐collected thoracic lymph (NC) group and a collected thoracic lymph (LC) group. Peripheral blood and thoracic lymph were collected from a jugular vein and a thoracic lymph duct, respectively, under anaesthesia. Bovine LF (bLF) was infused into the duodenal lumen by needle over a 1‐min period at a dose of 1 g kg−1. The transported bLF in the plasma and lymph was assayed quantitatively by double‐antibody enzyme‐linked immunosorbent assay (ELISA). Morphological investigation was also carried out in the intestine, lymph node, and liver. Following intraduodenal administration of bLF, the transported bLF in the NC group was detected in the plasma, and reached a peak value at 2 h. Furthermore, the bLF concentration in the thoracic duct lymph fluid in the LC group increased significantly, and peaked 2 h after the administration. In addition, bLF was not detected in the plasma of the LC group. Immunohistochemical analysis clearly showed anti‐bLF positive particles in the epithelial cells of the apical villi. The striated border and baso‐lateral membrane were also bLF positive. These results suggest that intraduodenally infused bLF is transported into the blood circulation via the lymphatic pathway, not via portal circulation in adult rats.
The electrophysiological and mechanical effects of 2,3‐butane‐dione monoxime and cytochalasin‐D in the Langendorff perfused rabbit heart
Authors: Kettlewell S. , Walker N. L. , Cobbe S. M. , Burton F. L. , Smith G. L.
Published: February 2004

Procedures that reduce contraction are used to facilitate optical measurements of membrane potential, but it is unclear to what extent they affect the excitability of the heart. This study has examined the electrophysiological consequences of a range of extracellular [Ca2+] (0.7–2.5 mmol l−1), 2,3‐butane‐dione monoxime (BDM; 1–20 mmol l−1) and cytochalasin‐D (Cyto‐D; 1–5 μmol l−1). Methods. Monophasic action potentials (MAPs) were recorded from the basal epicardial surface of the left ventricle of isolated rabbit hearts. Conduction delay (CD) and time to 90% repolarisation of the monophasic action potential (MAPD90) were measured. The effects of BDM and Cyto‐D on restitution were studied at a [Ca2+] of 1.9 mmol l−1. Restitution curves for MAPD90 were generated using a standard S1–S2 protocol. Results. All manoeuvres decreased left ventricular developed pressure (LVDP): 0.7 mmol l−1 Ca2+ to 74.0 ± 6.1%, 20 mmol l−1 BDM to 4.5 ± 1.0%, and 5 μmol l−1 Cyto‐D to 12.8 ± 3.5% of control value. CD decreased from a control value (33.3 ± 1.0 ms, n= 16) to 93.0 ± 2.2% in 0.7 mmol l−1 Ca2+, but increased to 133.7 ± 10.5% in 20 mmol l−1 BDM and 127.4 ± 10.6% in 5 μmol l−1 Cyto‐D. At 350 ms pacing cycle length, MAPD90 (control = 119.6 ± 1.7 ms n= 16) was prolonged by reduced extracellular [Ca2+]. BDM had no effects on MAPD90 at control pacing rates. Cyto‐D caused a significant prolongation (to 115.0 ± 3.0% of control, n= 6) at the highest concentration studied (5 μmol l−1). Both BDM (20 mmol l−1) and Cyto‐D (3 μmol l−1) flattened the restitution curves but neither agent altered maximum MAPD90. Conclusions. Extracellular [Ca2+] of 1.9 mmol l−1 in conjunction with a moderate dose of Cyto‐D (3 μmol l−1) reduced contractility with minimal effects on action potential duration and conduction at a fixed pacing cycle length. However, both BDM and Cyto‐D had pronounced effects on electrical restitution.
Phenotypic differences in cholinergic responses of distal colonic epithelium
Authors: Prior T. , Hernandez J. , Tougas G. , Rangachari P. K.
Published: February 2004

The Flinders sensitive line (FSL) rats exhibit an increased cholinergic responsiveness in vivo when compared to their counterparts, the Flinders resistant line (FRL) rats. The functional consequences of this phenotypic difference on colonic mucosal function are not known. We sought to determine whether isolated distal colonic mucosa from the two strains exhibit differential responses to cholinergic agonists. The responses of the distal colonic mucosa from two lines of rats to carbachol were compared by recording changes in short‐circuit current. The ion movements associated with these changes were assessed by flux analysis of the radiotracers, 22Na and 36Cl. The anticipated hyper‐responsiveness to cholinergic stimulation in FSL rats was not seen. Carbachol responses were significantly enhanced by indomethacin pretreatment only in FRL rats. Tetrodotoxin (TTX) pretreatment significantly reduced responses to carbachol in FSL rats at all concentrations tested, though this was only seen with lower concentrations in FRL rats. Flux analysis indicated that both lines absorbed Na+ and Cl− under basal conditions and that a significant residual flux was present. Stimulation with carbachol led to significant reductions in net Na+ and Cl− fluxes in both lines. The changes in net Na+ and Cl− flux in both lines stem largely from a decrease in mucosal to serosal fluxes of both ions with an increase in serosal to mucosal flux of Cl−. The striking difference is the significant reduction in residual flux seen only in FRL rats. Indomethacin pretreatment abolished the changes in residual flux seen in FRL rats. Thus the responses to carbachol in these rats had at least three components: (a) a direct effect on the transporting colonocyte, (b) an indirect effect mediated by an arachidonic acid metabolite, and (c) another indirect effect involving a neurotransmitter. The relative contributions of each of these components were different in the two lines.
Sand Fly Saliva EnhancesLeishmania amazonensisInfection by Modulating Interleukin-10 Production
Authors: Norsworthy Nilufer B. , Sun Jiaren , Elnaiem Dia , Lanzaro Gregory , Soong Lynn
Published: February 2004

After transmission through the bite of female sand flies,Leishmaniaspp. can cause a broad spectrum of disease manifestations collectively known as leishmaniases.L. amazonensisis endemic in South America, where it causes cutaneous, diffuse cutaneous, and visceral leishmaniasis. In this study, we have provided evidence that salivary gland extracts (SGE) ofLutzomyia longipalpisenhancesL. amazonensisinfection. BALB/c mice infected intradermally in the ear with 105metacyclic promastigotes ofL. amazonensistogether with SGE (equivalent to 0.5 gland) showed an early onset of disease and larger lesions that contained ∼3-log-units more parasites than did controls. To determine the potential mechanism underlying this enhancement, we assessed cytokine production via reverse transcriptase PCR and enzyme-linked immunosorbent assay. Mice coinjected with parasites and SGE displayed higher levels of interleukin-10 (IL-10) mRNA in the ear tissues, as well as higher levels of IL-10 in supernatants of restimulated draining lymph node (LN) cells, than did controls. Flow cytometric analysis revealed high frequencies of IL-10-producing CD4+and CD8+T cells in the draining LN of mice coinjected with the parasite and SGE. In addition, we examined bone marrow derived-macrophage cultures and detected increased IL-10 but decreased nitric oxide (NO) production in cells exposed to SGE prior to infection withL. amazonensis. Together, these results imply that the sand fly saliva facilitatesLeishmaniaevasion of the host immune system by modulating IL-10 production.
STAT1 Is Essential for Antimicrobial Effector Function but Dispensable for Gamma Interferon Production duringToxoplasma gondiiInfection
Authors: Gavrilescu L. Cristina , Butcher Barbara A. , Del Rio Laura , Taylor Gregory A. , Denkers Eric Y.
Published: February 2004

The opportunistic protozoanToxoplasma gondiiis a prototypic Th1-inducing pathogen inducing strong gamma interferon (IFN-γ) cytokine responses that are required to survive infection. Intracellular signaling intermediate STAT1 mediates many effects of IFN-γ and is implicated in activation of T-bet, a master regulator of Th1 differentiation. Here, we show thatT. gondii-infected STAT1-null mice fail to upregulate the IFN-γ-dependent effector molecules inducible nitric oxide synthase (iNOS), IGTP, and LRG-47, which are required for mice to survive infection. Both T-bet and interleukin-12 receptor β2 (IL-12Rβ2) failed to undergo normal upregulation in response toT. gondii. Development of IFN-γ-producing CD4+and CD8+T lymphocytes was severely curtailed in the absence of STAT1, but a substantial level of STAT1-independent non-T-cell-derived IFN-γ was induced. Absence of STAT1 also resulted in increased IL-4, Arg1, Ym1, and Fizz1, markers of Th2 differentiation and alternative macrophage activation. Together, the results show thatT. gondiiinduces STAT1-dependent T-lymphocyte and STAT1-independent non-T-cell IFN-γ production, but that effector functions of this type 1 cytokine cannot operate in the absence of STAT1, resulting in extreme susceptibility to acute infection.
Aerosolized Gamma Interferon (IFN-γ) Induces Expression of the Genes Encoding the IFN-γ-Inducible 10-Kilodalton Protein but Not Inducible Nitric Oxide Synthase in the Lung during Tuberculosis
Authors: Raju Bindu , Hoshino Yoshihiko , Kuwabara Kenichi , Belitskaya Ilana , Prabhakar Savita , Canova Antony , Gold Jeffrey A. , Condos Rany , Pine Richard I. , Brown Stuart , Rom William N. , Weiden Michael D.
Published: February 2004

Gamma interferon (IFN-γ) is critical in the immune response againstMycobacterium tuberculosis. In an ongoing trial of aerosol IFN-γ in conjunction with standard drug therapy, we have observed activation of IFN signaling in bronchoalveolar lavage (BAL) cells from tuberculosis (TB) patients. We hypothesized that aerosol IFN-γ treatment of pulmonary TB would increase expression of genes important for the control of TB. We investigated the expression of downstream genes by measuring inducible nitric oxide synthase (iNOS) and the chemokine IFN-inducible 10-kDa protein (IP-10) by real-time quantitative reverse transcription-PCR. In vitro,M. tuberculosisinduced IP-10, and IFN-γ stimulated this further, with no effect on iNOS expression. We studied 21 patients with pulmonary TB and 7 healthy subjects. Similar to the in vitro model, IP-10 mRNA was increased in BAL cells from TB patients and was augmented after treatment with aerosolized IFN-γ. TB was also associated with elevated iNOS mRNA, but aerosolized IFN-γ did not further enhance expression. Genomic analysis identified 1,300 of 4,058 genes expressed in BAL cells from six TB patients before and after 1 month of therapy, including aerosolized IFN-γ. However, only 15 genes were differentially regulated by IFN-γ. We conclude that iNOS and IP-10 mRNA expression is increased in TB but that aerosol IFN-γ treatment increases expression of few genes in the human lung.
Exogenous Gamma and Alpha/Beta Interferon Rescues Human Macrophages from Cell Death Induced byBacillus anthracis
Authors: Gold Jeffrey A. , Hoshino Yoshihiko , Hoshino Satomi , Jones Marcus B. , Nolan Anna , Weiden Michael D.
Published: February 2004

During the recent bioterrorism-related outbreaks, inhalational anthrax had a 45% mortality in spite of appropriate antimicrobial therapy, underscoring the need for better adjuvant therapies. The variable latency between exposure and development of disease suggests an important role for the host's innate immune response. Alveolar macrophages are likely the first immune cells exposed to inhalational anthrax, and the interferon (IFN) response of these cells comprises an important arm of the host innate immune response to intracellular infection withBacillus anthracis. Furthermore, IFNs have been used as immunoadjuvants for treatment of another intracellular pathogen,Mycobacterium tuberculosis. We established a model ofB. anthracisinfection with the Sterne strain (34F2) which contains lethal toxin (LeTx). 34F2was lethal to murine and human macrophages. Treatment with IFNs significantly improved cell viability and reduced the number of germinated intracellular spores. Infection with 34F2failed to induce the latent transcription factors signal transducer and activators of transcription 1 (STAT1) and ISGF-3, which are central to the IFN response. Furthermore, 34F2reduced STAT1 activation in response to exogenous alpha/beta IFN, suggesting direct inhibition of IFN signaling. Even though 34F2has LeTx, there was no mitogen-activated protein kinase kinase 3 cleavage and p38 was normally induced, suggesting that these early effects ofB. anthracisinfection in macrophages are independent of LeTx. These data suggest an important role for both IFNs in the control ofB. anthracisand the potential benefit of using exogenous IFN as an immunoadjuvant therapy.
Organ Injury and Cytokine Release Caused by Peptidoglycan Are Dependent on the Structural Integrity of the Glycan Chain
Authors: Myhre Anders E. , Stuestøl Jon Fredrik , Dahle Maria K. , Øverland Gunhild , Thiemermann Christoph , Foster Simon J. , Lilleaasen Per , Aasen Ansgar O. , Wang Jacob E.
Published: February 2004

Several studies have implicated a role of peptidoglycan (PepG) as a pathogenicity factor in sepsis and organ injury, in part by initiating the release of inflammatory mediators. We wanted to elucidate the structural requirements of PepG to trigger inflammatory responses and organ injury. Injection of native PepG into anesthetized rats caused moderate but significant increases in the levels of alanine aminotransferase, aspartate aminotransferase, γ-glutamyl transferase, and bilirubin (markers of hepatic injury and/or dysfunction) and creatinine and urea (markers of renal dysfunction) in serum, whereas PepG pretreated with muramidase to digest the glycan backbone failed to do this. In an ex vivo model of human blood, PepG containing different amino acids induced similar levels of the cytokines tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), IL-8, and IL-10, as determined by plasma analyses (enzyme-linked immunosorbent assay). Hydrolysis of theStaphylococcus aureuscross-bridge with lysostaphin resulted in moderately reduced release of TNF-α, IL-6, IL-8, and IL-10, whereas muramidase digestion nearly abolished the ability to induce cytokine release and IL-6 mRNA accumulation in CD14+monocytes compared to intact PepG. However, additional experiments showed that muramidase-treated PepG synergized with lipopolysaccharide to induce TNF-α and IL-10 release in whole blood, despite its lack of inflammatory activity when administered alone. Based on these studies, we hypothesize that the structural integrity of the glycan chain of the PepG molecule is very important for the pathogenic effects of PepG. The amino acid composition of PepG, however, does not seem to be essential for the inflammatory properties of the molecule.
Biological Activities ofBacteroides forsythusLipoproteins and Their Possible Pathological Roles in Periodontal Disease
Authors: Hasebe Akira , Yoshimura Atsutoshi , Into Takeshi , Kataoka Hideo , Tanaka Saori , Arakawa Shinichi , Ishikura Hiroaki , Golenbock Douglas T. , Sugaya Tsutomu , Tsuchida Nobuo , Kawanami Masamitsu , Hara Yoshitaka , Shibata Ken-ichiro
Published: February 2004

Bacteroides forsythusis a gram-negative, anaerobic, fusiform bacterium and is considered to be an etiological agent in periodontal disease. A lipoprotein fraction prepared fromB. forsythuscells by Triton X-114 phase separation (BfLP) activated human gingival fibroblasts and a human monocytic cell line, THP-1, to induce interleukin-6 production and tumor necrosis factor alpha production. BfLP was found to be capable of inducing nuclear factor-κB translocation in human gingival fibroblasts and THP-1 cells. By using Chinese hamster ovary K1 cells transfected with Toll-like receptor genes together with a nuclear factor-κB-dependent CD25 reporter plasmid, it was found that signaling by BfLP was mediated by Toll-like receptor 2 but not by CD14 or Toll-like receptor 4. BfLP induced apoptotic cell death in human gingival fibroblasts, KB cells (an oral epithelial cell line), HL-60 cells (a human myeloid leukemia cell line), and THP-1 cells but not in MOLT4 cells (a T-cell leukemia cell line). Caspase-8, an initiator caspase in apoptosis, was found to be activated in these cells in response to BfLP stimulation. Thus, this study suggested that BfLP plays some etiological roles in oral infections, especially periodontal disease, by induction of cell activation or apoptosis.
Glyceraldehyde-3-Phosphate Dehydrogenase of
Streptococcus oralis
Functions as a Coadhesin for
Porphyromonas gingivalis
Major Fimbriae
Authors: Maeda Kazuhiko , Nagata Hideki , Yamamoto Yumiko , Tanaka Muneo , Tanaka Junko , Minamino Naoto , Shizukuishi Satoshi
Published: February 2004

Cohesive interactions between
Porphyromonas gingivalis
and plaque-forming bacteria, such as
Streptococcus oralis
, are considered to play an important role in the colonization of
P. gingivalis
in periodontal sites. Although
P. gingivalis
fimbriae have been reported to mediate coaggregation with
S. oralis
, the
S. oralis
molecule involved has not been identified. We identified the coadhesin of
S. oralis
ATCC 9811 and purified it by affinity column chromatography. We found that the molecular mass of the purified protein was approximately 40 kDa. Dot blot and Western blot assays showed binding of the 40-kDa protein to
P. gingivalis
fimbriae. Further, turbidimetric assays showed that the coadhesin inhibited coaggregation between
P. gingivalis
and
S. oralis
in a dose-dependent manner. Analyses of the amino-terminal sequences of the protein and its lysyl endopeptidase-cleaved fragments revealed that the coadhesin was identical to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Next, we cloned the gene that encodes
S. oralis
GAPDH and found that the sequence had a high degree of homology with the sequences of GAPDHs of various bacteria, including
Streptococcus gordonii
and
Fusobacterium nucleatum
. To confirm the contribution of
S. oralis
GAPDH to the interaction with
P. gingivalis
, a recombinant GAPDH protein was generated in
Escherichia coli
; this protein bound to
P. gingivalis
fimbriae and had an inhibitory effect on coaggregation. These results suggest that
S. oralis
GAPDH functions as a coadhesin for
P. gingivalis
fimbriae. In addition, considering the high degree of homology of the GAPDHs of various bacteria, those of other plaque-forming bacteria also may contribute to the colonization of
P. gingivalis
.
Effect of Neutralizing Transforming Growth Factor β1 on the Immune Response againstMycobacterium tuberculosisin Guinea Pigs
Authors: Allen Shannon Sedberry , Cassone Lynne , Lasco Todd M. , McMurray David N.
Published: February 2004

Transforming growth factor β (TGF-β) is a cytokine which has been shown to suppress the antimycobacterial immune responses of humans and experimental animals. In this study, the contributions of TGF-β to cytokine production in vivo were investigated by using the established guinea pig model of tuberculous pleurisy.Mycobacterium bovisBCG-vaccinated guinea pigs were injected intrapleurally with heat-killed virulentMycobacterium tuberculosis. Eight days following induction of an antigen-specific pleural effusion, guinea pigs were injected intrapleurally with anti-TGF-β1 or isotype control antibody. The following day, pleural exudates were removed, and the fluid volume and characteristics of the infiltrating cells were determined. Pleural fluid was analyzed for total interferon (IFN) and tumor necrosis factor (TNF) protein levels by using appropriate bioassays. RNA from pleural effusion cells was examined to determine TGF-β1, TNF-α, IFN-γ, and interleukin-8 mRNA levels by using real-time PCR. Proliferative responses of pleural effusion lymphocytes were examined in response to concanavalin A and purified protein derivative (PPD) in vitro. Treatment with anti-TGF-β1 resulted in decreased pleural fluid volume and decreased cell numbers in the pleural space along with an increased percentage of lymphocytes and a decreased percentage of neutrophils. The bioactive TNF protein levels in pleural fluid were increased in guinea pigs treated with anti-TGF-β1, while the bioactive IFN protein concentrations were not altered. Expression of TGF-β1 and TNF-α mRNA was significantly increased following TGF-β1 neutralization. Finally, PPD-induced proliferative responses of pleural cells from anti-TGF-β1-treated animals were significantly enhanced. Thus, TGF-β1 may be involved in the resolution of this local, mycobacterial antigen-specific inflammatory response.
Humoral Responses to
Porphyromonas gingivalis
Gingipain Adhesin Domains in Subjects with Chronic Periodontitis
Authors: Nguyen Ky-Anh , DeCarlo Arthur A. , Paramaesvaran Mayuri , Collyer Charles A. , Langley David B. , Hunter Neil
Published: February 2004

The gingipains have been implicated in the pathogenicity of
Porphyromonas gingivalis
, a major etiologic agent of chronic periodontitis. Mature gingipains often present as a membrane-bound glycosylated proteinase-adhesin complex comprising multiple adhesin domains (HA1 to -4) and a catalytic domain. Using recombinant adhesin domains, we were able to show that patients with chronic periodontitis produce significantly more immunoglobulin G reactive with gingipain domains than a corresponding group with healthy periodontium. Titers were predominantly directed toward the carbohydrate epitopes shared between the gingipains and the lipopolysaccharide of
P. gingivalis
with little recognition of the peptide backbone of the catalytic domains. Distribution of titers to peptide epitopes of the adhesin domains was as follows: HA4 ≈ HA1 > HA3 ≫ HA2. No correlation was observed between markers of disease severity and titers to individual adhesins within the disease group. Posttreatment titers showed no change or a decrease in titers for the majority of patients except for titers to the HA2 domain which showed marked increases in a few responding patients. Since the HA2 domain is important in hemoglobin binding and acquisition of essential porphyrin, boosting titers of antibodies to this domain may have the potential to control the growth of this organism.
Role of NK Cells and Gamma Interferon in Transplacental Passage ofToxoplasma gondiiin a Mouse Model of Primary Infection
Authors: Abou-Bacar Ahmed , Pfaff Alexander W. , Georges Sophie , Letscher-Bru Valérie , Filisetti Denis , Villard Odile , Antoni Elisabeth , Klein Jean-Paul , Candolfi Ermanno
Published: February 2004

Protective immunity in mice infected withToxoplasma gondiiis mainly mediated by NK cells, CD4 and CD8 T cells, and type 1 cytokines, such as gamma interferon (IFN-γ). To clarify the roles of NK cells and IFN-γ in protection against primary congenital toxoplasmosis, we used recombination activating gene 2 knockout (RAG-2−/−) mice, which lack T and B lymphocytes, in comparison with the wild-type BALB/c model. RAG-2−/−mice had a significantly lower risk of fetal toxoplasmosis than BALB/c mice (25 versus 63.9%;P= 0.003). This protection was associated with an increased number of maternal NK cells, IFN-γ secretion by spleen cells, and decreased parasitemia. In the RAG-2−/−mice, NK cell depletion increased both the rate of fetal infection, to 56.5% (P= 0.02), and the blood parasite burden. Conversely, in the BALB/c mice, this treatment did not modify maternofetal transmission or the blood parasite burden. Neutralization of IFN-γ in both infected RAG-2−/−and BALB/c mice decreased congenitalToxoplasmatransmission, contrasting with an exacerbation of maternal infection. These data suggest that a partially protective immunity against congenital toxoplasmosis is achieved due to the increased number of NK cells in RAG-2−/−mice. However, it seems that IFN-γ enhances, directly or indirectly, the transplacental transmission.
Survival of
Aspergillus fumigatus
in Serum Involves Removal of Iron from Transferrin: the Role of Siderophores
Authors: Hissen A. H. T. , Chow J. M. T. , Pinto L. J. , Moore M. M.
Published: February 2004

Aspergillus fumigatus
is a filamentous fungus which can cause invasive disease in immunocompromised individuals.
A. fumigatus
can grow in medium containing up to 80% human serum, despite very low concentrations of free iron. The purpose of this study was to determine the mechanism by which
A. fumigatus
obtains iron from the serum iron-binding protein transferrin. In iron-depleted minimal essential medium (MEM),
A. fumigatus
growth was supported by the addition of holotransferrin (holoTf) or FeCl
3
but not by the addition of apotransferrin (apoTf). Proteolytic degradation of transferrin by
A. fumigatus
occurred in MEM-serum; however, transferrin degradation did not occur until late logarithmic phase. Moreover, transferrin was not degraded by
A. fumigatus
incubated in MEM-holoTf. Urea polyacrylamide gel electrophoresis showed that in MEM-holoTf, holoTf was completely converted to apoTf by
A. fumigatus
. In human serum, all of the monoferric transferrin was converted to apoTf within 8 h. Siderophores were secreted by
A. fumigatus
after 8 h of growth in MEM-serum and 12 h in MEM-holoTf. The involvement of small molecules in iron acquisition was confirmed by the fact that transferrin was deferrated by
A. fumigatus
even when physically separated by a 12-kDa-cutoff membrane. Five siderophores were purified from
A. fumigatus
culture medium, and the two major siderophores were identified as triacetylfusarinine C and ferricrocin. Both triacetylfusarinine C and ferricrocin removed iron from holoTf with an affinity comparable to that of ferrichrome. These data indicate that
A. fumigatus
survival in human serum in vitro involves siderophore-mediated removal of iron from transferrin. Proteolytic degradation of transferrin may play a secondary role in iron acquisition.
The
Klebsiella pneumoniae
O Antigen Contributes to Bacteremia and Lethality during Murine Pneumonia
Authors: Shankar-Sinha Sunita , Valencia Gabriel A. , Janes Brian K. , Rosenberg Jessica K. , Whitfield Chris , Bender Robert A. , Standiford Ted J. , Younger John G.
Published: February 2004

Bacterial surface carbohydrates are important pathogenic factors in gram-negative pneumonia infections. Among these factors, O antigen has been reported to protect pathogens against complement-mediated killing. To examine further the role of O antigen, we insertionally inactivated the gene encoding a galactosyltransferase necessary for serotype O1 O-antigen synthesis (
wbbO
) from
Klebsiella pneumoniae
43816. Analysis of the mutant lipopolysaccharide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed the absence of O antigen. In vitro, there were no detectable differences between wild-type
K. pneumoniae
and the O-antigen-deficient mutant in regard to avid binding by murine complement C3 or resistance to serum- or whole-blood-mediated killing. Nevertheless, the 72-h 50% lethal dose of the wild-type strain was 30-fold greater than that of the mutant (2 × 10
3
versus 6 × 10
4
CFU) after intratracheal injection in ICR strain mice. Despite being less lethal, the mutant organism exhibited comparable intrapulmonary proliferation at 24 h compared to the level of the wild type. Whole-lung chemokine expression (CCL3 and CXCL2) and bronchoalveolar inflammatory cell content were also similar between the two infections. However, whereas the wild-type organism produced bacteremia within 24 h of infection in every instance, bacteremia was not seen in mutant-infected mice. These results suggest that during murine pneumonia caused by
K. pneumoniae
, O antigen contributes to lethality by increasing the propensity for bacteremia and not by significantly changing the early course of intrapulmonary infection.
Bordetella pertussisInfection of Primary Human Monocytes Alters HLA-DR Expression
Authors: Shumilla Jennifer A. , Lacaille Vashti , Hornell Tara M. C. , Huang Jennifer , Narasimhan Supraja , Relman David A. , Mellins Elizabeth D.
Published: February 2004

Bordetella pertussisis the causative agent of whooping cough, a potentially lethal respiratory disease in children. In immunocompetent individuals,B. pertussisinfection elicits an effective adaptive immune response driven by activated CD4+T cells. However, liveB. pertussispersists in the host for 3 to 4 weeks prior to clearance. Thus,B. pertussisappears to have evolved short-term mechanisms for immune system evasion. We investigated the effects ofB. pertussiswild-type strain BP338 on antigen presentation in primary human monocytes. BP338 infection reduced cell surface expression of HLA-DR and CD86 but not that of major histocompatibility complex class I proteins. This change in cell surface HLA-DR expression reflected intracellular redistribution of HLA-DR. The proportion of peptide-loaded molecules was unchanged in infected cells, suggesting that intracellular retention occurred after peptide loading. AlthoughB. pertussisinfection of monocytes induced rapid and robust expression of interleukin-10 (IL-10), HLA-DR redistribution did not appear to be explained by increased IL-10 levels. BP338-infected monocytes exhibited reduced synthesis of HLA-DR dimers. Interestingly, those HLA-DR proteins that were generated appeared to be longer-lived than HLA-DR in uninfected monocytes. BP338 infection also prevented gamma interferon (IFN-γ) induction of HLA-DR protein synthesis. Using mutant strains ofB. pertussis, we found that reduction in HLA-DR surface expression was due in part to the presence of pertussis toxin whereas the inhibition of IFN-γ induction of HLA-DR could not be linked to any of the virulence factors tested. These data demonstrate thatB. pertussisutilizes several mechanisms to modulate HLA-DR expression.
Cross-Species Surface Display of Functional Spirochetal Lipoproteins by Recombinant
Borrelia burgdorferi
Authors: Zückert Wolfram R. , Lloyd Jill E. , Stewart Philip E. , Rosa Patricia A. , Barbour Alan G.
Published: February 2004

Surface-exposed lipoproteins of relapsing fever (RF) and Lyme borreliosis
Borrelia
spirochetes mediate certain interactions of the bacteria with their arthropod and vertebrate hosts. RF spirochetes such as
Borrelia hermsii
serially evade the host's antibody response by multiphasic antigenic variation of Vsp and Vlp proteins. Furthermore, the expression of Vsp1 and Vsp2 by
Borrelia turicatae
is associated with neurotropism and higher blood densities, respectively. In contrast to RF
Borrelia
species, the Lyme borreliosis spirochete
Borrelia burgdorferi
is amenable to genetic manipulation. To facilitate structure-function analyses of RF surface lipoproteins, we used recombinant plasmids to introduce full-length
vsp1
and
vsp2
as well as two representative
vlp
genes into
B. burgdorferi
cells. Recombinant
B. burgdorferi
cells constitutively expressed the proteins under the control of the
B. burgdorferi flaB
promoter. Antibody and protease accessibility assays indicated proper surface exposure and folding. Expression of Vsp1 and Vsp2 conferred glycosaminoglycan binding to recombinant
B. burgdorferi
cells that was similar to that observed with purified recombinant proteins and
B. turicatae
expressing native Vsp. These data demonstrate that the lipoprotein modification and export mechanisms in the genus
Borrelia
are conserved. They also validate the use of recombinant
B. burgdorferi
in studies of surface lipoprotein structure-function and the biogenesis of spirochete membranes.
Nutritional Effects on Host Response to Lung Infections with Mucoid
Pseudomonas aeruginosa
in Mice
Authors: van Heeckeren Anna M. , Schluchter Mark , Xue Lintong , Alvarez Juan , Freedman Steven , St. George Judith , Davis Pamela B.
Published: February 2004

In cystic fibrosis, a recessive genetic disease caused by defects in the cystic fibrosis conductance regulator (CFTR), the main cause of death is lung infection and inflammation. Nutritional deficits have been proposed to contribute to the excessive host inflammatory response in both humans and
Cftr
-knockout mice.
Cftr
-knockout mice and gut-corrected
Cftr
-knockout mice expressing human CFTR primarily in the gut were challenged with
Pseudomonas aeruginosa
-laden agarose beads; they responded similarly with respect to bronchoalveolar lavage cell counts and levels of the acute-phase cytokines tumor necrosis factor alpha, interleukin-1β (IL-1β), and IL-6. Wild-type mice fed the liquid diet used to prevent intestinal obstruction in
Cftr
-knockout mice had inflammatory responses to
P. aeruginosa
-laden agarose beads similar to those of wild-type mice fed an enriched solid diet, so dietary effects are unlikely to account for differences between wild-type mice and mice with cystic fibrosis. Finally, since cystic fibrosis patients and
Cftr
-knockout mice have an imbalance in fatty acids (significantly lower-than-normal levels of docosahexaenoic acid), the effects of specific supplementation with docosahexaenoic acid of wild-type and
Cftr
-knockout mice on their inflammatory responses to
P. aeruginosa
-laden agarose beads were tested. There were no significant differences (
P
= 0.35) in cumulative survival rates between
Cftr
-knockout mice and wild-type mice provided with either the liquid diet Peptamen or Peptamen containing docosahexaenoic acid. In conclusion, diet and docosahexaenoic acid imbalances alone are unlikely to explain the differences in the host response to lung infections with mucoid
P. aeruginosa
between mice with cystic fibrosis and their wild-type counterparts.
Nod1 Is an Essential Signal Transducer in Intestinal Epithelial Cells Infected with Bacteria That Avoid Recognition by Toll-Like Receptors
Authors: Kim Jae Gyu , Lee Sung Joong , Kagnoff Martin F.
Published: February 2004

The transcription factor NF-κB in human intestinal epithelial cells plays a central role in regulating genes that govern the onset of mucosal inflammatory responses following intestinal microbial infection. Nod1 is a cytosolic pattern recognition receptor in mammalian cells that senses components of microbial peptidoglycans and signals the activation of NF-κB. The aim of these studies was to assess the functional importance of Nod1 in activating NF-κB and NF-κB proinflammatory target genes in human intestinal epithelium. Human colon epithelial cells that constitutively express Nod1 were used as a model intestinal epithelium. These cells do not signal through Toll-like receptor 4 (TLR4) or respond to bacterial lipopolysaccharide, but they express functional TLR5 and interleukin 1 (IL-1) receptors that signal the activation of NF-κB in response to bacterial flagellin or IL-1 stimulation. Stable expression of dominant negative (DN) Nod1 in colon epithelial cells prevented IκB kinase and NF-κB activation in response to infection with enteroinvasiveEscherichia coli. In contrast, DN Nod1 did not eliminate IL-1 or flagellin-stimulated NF-κB activation. Inhibition of NF-κB was accompanied by inhibition of NF-κB target genes that provide signals for the mucosal influx of neutrophils during intestinal infection. We conclude that signaling through Nod1 is required for activating NF-κB in human intestinal epithelial cells infected with gram-negative enteric bacteria that can bypass TLR activation. Signaling through Nod1 provides the intestinal epithelium with a backup mechanism for rapidly activating innate immunity during infection with a group of highly invasive pathogenic gram-negative bacteria.
Evidence for a Hybrid Genomic Island in Verocytotoxin-Producing
Escherichia coli
CL3 (Serotype O113:H21) Containing Segments of EDL933 (Serotype O157:H7) O Islands 122 and 48
Authors: Shen Songhai , Mascarenhas Mariola , Rahn Kris , Kaper James B. , Karmal Mohamed A.
Published: February 2004

Genomic O island 122 (OI-122) of the verocytotoxin-producing
Escherichia coli
(VTEC) strain EDL933 contains four putative virulence genes, Z4321, Z4326, Z4332, and Z4333. However, strain CL3 (serotype O113:H21) contains only Z4321, not the other three genes. To determine whether Z4321 is part of a different genomic island in CL3, a region of 27,293 bp up- and downstream of Z4321 was sequenced and found to contain elements of two different EDL933 genomic islands (OI-48 and OI-122) and a
Yersinia pestis
-like hemolysin/adhesin gene cluster. The region contained OI-48 genes Z1635, Z1636, and Z1637 at the left terminus and Z1641, Z1642, Z1643, and Z1644 at the right. The middle portion consisted of OI-48 gene Z1640, which was separated into three fragments by genomic segments including the
Y. pestis
cluster and EDL933 OI-122 genes Z4322, Z4321, and Z4318. In a PCR investigation of 36 VTEC strains of different serotypes, intact Z1640 was present in strains of serotypes O157:H7, O26:H11, O103:H2, O111:NM, and O145:NM, which are associated with hemolytic uremic syndrome and outbreaks. In contrast, fragmented Z1640 was seen in strains of nonepidemic serotypes, such as O91:H21 and O113:H21, and in animal serotypes that have not been associated with human disease, indicating that Z1640 might be a virulence gene.
Helicobacter pylori
and Complex Gangliosides
Authors: Roche Niamh , Ångström Jonas , Hurtig Marina , Larsson Thomas , Borén Thomas , Teneberg Susann
Published: February 2004

Recognition of sialic acid-containing glycoconjugates by the human gastric pathogen
Helicobacter pylori
has been repeatedly demonstrated. To investigate the structural requirements for
H. pylori
binding to complex gangliosides, a large number of gangliosides were isolated and characterized by mass spectrometry and proton nuclear magnetic resonance. Ganglioside binding of sialic acid-recognizing
H. pylori
strains (strains J99 and CCUG 17874) and knockout mutant strains with the sialic acid binding adhesin SabA or the NeuAcα3Galβ4GlcNAcβ3Galβ4GlcNAcβ-binding neutrophil-activating protein HPNAP deleted was investigated using the thin-layer chromatogram binding assay. The wild-type bacteria bound to
N
-acetyllactosamine-based gangliosides with terminal α3-linked NeuAc, while gangliosides with terminal NeuGcα3, NeuAcα6, or NeuAcα8NeuAcα3 were not recognized. The factors affecting binding affinity were identified as (i) the length of the
N
-acetyllactosamine carbohydrate chain, (ii) the branches of the carbohydrate chain, and (iii) fucose substitution of the
N
-acetyllactosamine core chain. While the J99/NAP
−
mutant strain displayed a ganglioside binding pattern identical to that of the parent J99 wild-type strain, no ganglioside binding was obtained with the J99/SabA
−
mutant strain, demonstrating that the SabA adhesin is the sole factor responsible for the binding of
H. pylori
bacterial cells to gangliosides.
Adenylate Cyclase Toxin fromBordetella pertussisSynergizes with Lipopolysaccharide To Promote Innate Interleukin-10 Production and Enhances the Induction of Th2 and Regulatory T Cells
Authors: Ross Pádraig J. , Lavelle Ed C. , Mills Kingston H. G. , Boyd Aoife P.
Published: February 2004

Adenylate cyclase toxin (CyaA) fromBordetella pertussiscan subvert host immune responses allowing bacterial colonization. Here we have examined its adjuvant and immunomodulatory properties and the possible contribution of lipopolysaccharide (LPS), known to be present in purified CyaA preparations. CyaA enhanced antigen-specific interleukin-5 (IL-5) and IL-10 production and immunoglobulin G1 antibodies to coadministered antigen in vivo. Antigen-specific CD4+-T-cell clones generated from mice immunized with antigen and CyaA had cytokine profiles characteristic of Th2 or type 1 regulatory T (Tr1) cells. Since innate immune cells direct the induction of T-cell subtypes, we examined the influence of CyaA on activation of dendritic cells (DC) and macrophages. CyaA significantly augmented LPS-induced IL-6 and IL-10 and inhibited LPS-driven tumor necrosis factor alpha and IL-12p70 production from bone marrow-derived DC and macrophages. CyaA also enhanced cell surface expression of CD80, CD86, and major histocompatibility class II on immature DC. The stimulatory activity of our CyaA preparation for IL-10 production and CD80, CD86, and major histocompatibility complex class II expression was attenuated following the addition of polymyxin B or with the use of DC from Toll-like receptor (TLR) 4-defective mice. However, treatment of DC with LPS alone at the concentration present in the CyaA preparation (0.2 ng/ml) failed to activate DC in vitro. Our findings demonstrate that activation of innate cells in vitro by CyaA is dependent on a second signal through a TLR and that CyaA can promote Th2/Tr1-cell responses by inhibiting IL-12 and promoting IL-10 production by DC and macrophages.
Dissolved Oxygen Levels Alter Gene Expression and Antigen Profiles in
Borrelia burgdorferi
Authors: Seshu J. , Boylan Julie A. , Gherardini Frank C. , Skare Jonathan T.
Published: February 2004

The Lyme disease spirochete,
Borrelia burgdorferi
, encounters many environmental signals as it cycles between the arthropod vector and mammalian hosts, including temperature, pH, and other host factors. To test the possibility that dissolved oxygen modulates gene expression in
B. burgdorferi
, spirochetes were exposed to differential levels of dissolved oxygen, and distinct alterations were observed at both the transcriptional and translational levels. Specifically NapA, a Dps/Dpr homologue involved in the oxidative stress response in other bacteria, was reduced when
B. burgdorferi
was grown under oxygen-limiting conditions. In contrast, several immunoreactive proteins were altered when tested with infection-derived sera from different hosts. Specifically, OspC, DbpA, and VlsE were synthesized at greater levels when cells were grown in limiting oxygen, whereas VraA was reduced. The levels of oxygen in the medium did not affect OspA production. Real-time reverse transcription-PCR analysis of RNA isolated from infectious isolates of strains B31 and cN40 indicated that the expression of
ospC
,
dbpA
, and
vlsE
increased while
napA
expression decreased under dissolved-oxygen-limiting conditions, whereas
flaB
was not affected. The reverse transcription-PCR results corroborated the immunoblot analyses and indicated that the increase in OspC, DbpA, and VlsE was due to regulation at the transcriptional level of the genes encoding these antigens. These results indicate that dissolved oxygen modulates gene expression in
B. burgdorferi
and imply that the redox environment may be an additional regulatory cue that spirochetes exploit to adapt to the disparate niches that they occupy in nature.
Role for Serine Protease HtrA (DegP) ofStreptococcus pyogenesin the Biogenesis of Virulence Factors SpeB and the Hemolysin Streptolysin S
Authors: Lyon William R. , Caparon Michael G.
Published: February 2004

The serine protease HtrA is involved in the folding and maturation of secreted proteins, as well as in the degradation of proteins that misfold during secretion. Depletion of HtrA has been shown to affect the sensitivity of many organisms to thermal and environmental stresses, as well as being essential for virulence in many pathogens. In the present study, we compared the behaviors of several different HtrA mutants of the gram-positive pathogenStreptococcus pyogenes(group A streptococcus). Consistent with prior reports, insertional inactivation ofhtrA, the gene that encodes HtrA, resulted in a mutant that grew poorly at 37°C. However, an identical phenotype was observed when a similar polar insertion was placed immediately downstream ofhtrAin the streptococcal chromosome, suggesting that the growth defect of the insertion mutant was not a direct result of insertional inactivation ofhtrA. This conclusion was supported by the observation that a nonpolar deletion mutation ofhtrAdid not produce the growth defect. However, this mutation did affect the production of several secreted virulence factors whose biogenesis requires extensive processing. For the SpeB cysteine protease, the loss of HtrA was associated with a failure to proteolytically process the zymogen to an active protease. For the streptolysin S hemolysin, a dramatic increase in hemolytic activity resulted from the depletion of HtrA. Interestingly, HtrA-deficient mutants were not attenuated in a murine model of subcutaneous infection. These data add to the growing body of information that implies an important role for HtrA in the biogenesis of secreted proteins in gram-positive bacteria.
An Adenylate Cyclase-Controlled Signaling Network Regulates
Pseudomonas aeruginosa
Virulence in a Mouse Model of Acute Pneumonia
Authors: Smith Roger S. , Wolfgang Matthew C. , Lory Stephen
Published: February 2004

Infections caused by the opportunistic pathogen
Pseudomonas aeruginosa
involve the interplay of several bacterial virulence factors. It has recently been established that the delivery of toxic effector proteins by the type III secretion system is an important virulence mechanism in several animal models. Furthermore, the expression of the type III secretion system and its effectors has been correlated with a poor clinical outcome during human infections. A novel cyclic AMP (cAMP) regulatory network that controls the expression of virulence factors, including the type III secretion system, was examined to determine its contribution to
P. aeruginosa
colonization and dissemination in a mouse pneumonia model. Mutants lacking the two genome-encoded adenylate cyclases, CyaA and CyaB, and the cAMP-dependent regulator Vfr were examined. Based on the enumeration of bacteria in lungs, livers, and spleens, as well as the assessment of mouse lung pathology, mutations in the
cyaB
and
vfr
genes resulted in a more significantly attenuated phenotype than mutations in
cyaA
. Moreover, in this model, expression of the type III secretion system was essential for lung colonization and pathology. Strains with mutations in the
exsA
gene, which encodes a type III regulatory protein, or
pscC
, which encodes an essential component of the secretion apparatus, were also significantly attenuated. Finally, we demonstrate that virulence can be restored in an adenylate cyclase mutant by the overexpression of
exsA
, which specifically restores expression of the type III secretion system in the absence of a functional cAMP-dependent regulatory network.
CNLAC1Is Required for Extrapulmonary Dissemination ofCryptococcus neoformansbut Not Pulmonary Persistence
Authors: Noverr Mairi C. , Williamson Peter R. , Fajardo Ryan S. , Huffnagle Gary B.
Published: February 2004

The pathogenic yeastCryptococcus neoformansproduces a laccase enzyme (CNLAC1), which catalyzes the synthesis of melanin in the presence of phenolic compounds. A number of genes have been implicated in the regulation of laccase and melanization, includingIPC1,GPA1,MET3, andSTE12. Albino mutants derived from random mutagenesis techniques may contain mutations in genes that regulate multiple virulence factors, includingCNLAC1. The goal of our study is to investigate the role ofCNLAC1in virulence and evasion of pulmonary host defenses after infection via the respiratory tract. Using a set of congenic laccase-positive (2E-TUC-4) and laccase-deficient (2E-TU-4) strains, we found that both strains are avirulent at a lower dose (104CFU/mouse) in mice. After the infectious dose was increased to 106CFU/mouse, 70% mortality was observed in mice infected with 2E-TUC-4 compared to no mortality in mice infected with 2E-TU-4 at day 30 postinfection. This observation confirms the requirement forCNLAC1in virulence. Interestingly, we observed no differences between the two strains in pulmonary growth or in elicitation of cellular immune responses in the lung. The only measurable defect of 2E-TU-4 was in dissemination to extrapulmonary sites. To examine the role ofCNLAC1in dissemination, mice were infected intravenously. By week 3 postinfection, equal numbers of strains 2E-TUC-4 and 2E-TU-4 were recovered from the brain and spleen. This observation indicates thatCNLAC1facilitates escape from the lung, but not growth in the lungs or brain, and suggests a novel role forCNLAC1in virulence during an infection aquired via the respiratory tract.