bluuebunny/crossref_metadata_2025 huggingface dataset:
Association of
Lactobacillus
spp. with Peyer's Patches in Mice
Authors: Plant Laura , Conway Patricia
Published: July 2002

Sixteen strains of
Lactobacillus
isolated from humans, mice, and food products were screened for their capacity to associate with Peyer's patches in mice. In preliminary experiments, in vitro binding to tissue pieces was assessed by scanning electron microscopy, and it was demonstrated qualitatively that 5 of the 16 strains showed some affinity for the Peyer's patches, irrespective of their association with the nonlymphoid intestinal tissue.
Lactobacillus fermentum
KLD was selected for further study, since, in addition to its intrinsically high adhesion rate, this organism was found to exhibit a preferential binding to the follicle-associated epithelium of the Peyer's patches compared with its level of binding to the mucus-secreting regions of the small intestine. Quantitative assessment of scanning electron micrographs of tissue sections which had been incubated with
L. fermentum
KLD or a nonbinding control strain,
Lactobacillus delbruckii
subsp.
bulgaricus
, supported these observations, since a marked difference in adhesion was noted (
P
< 0.05). This preferential association of strain KLD with the Peyer's patches was also confirmed with radiolabeled lactobacilli incubated with intestinal tissue in the in vitro adhesion assay. Direct recovery of
L. fermentum
KLD from washed tissue following oral dosing of mice revealed a distinct association (
P
< 0.05) between this organism and the Peyer's patch tissue. In contrast,
L. delbruckii
subsp.
bulgaricus
showed negligible binding to both tissue types in both in vitro and in vivo adhesion assays. It was concluded that
L. fermentum
KLD bound preferentially to Peyer's patches of BALB/c mice.
PCR-Based Method for Isolation and Detection of
Chlamydia pneumoniae
DNA in Cerebrospinal Fluids
Authors: Ikejima Hideaki , Haranaga Shusaku , Takemura Hiromu , Kamo Tsutomu , Takahashi Youichi , Friedman Herman , Yamamoto Yoshimasa
Published: July 2002

Since current studies indicate the possible involvement of
Chlamydia pneumoniae
in the pathogenesis of multiple sclerosis (MS), demonstration of
C. pneumoniae
in the cerebrospinal fluid (CSF) of patients with MS is highly desirable. However, there is controversy concerning the detection of
C. pneumoniae
in CSFs from MS patients due to the lack of a standard protocol for extraction and detection of
C. pneumoniae
DNA. In this regard, we attempted to establish a highly effective extraction protocol for
C. pneumoniae
DNA from CSFs utilizing a commercial kit and a PCR detection method. The extraction and PCR detection protocol established in this study succeeded in detecting as few as 20
C. pneumoniae
organisms in 200 μl of mock CSF. The use of this protocol to detect
C. pneumoniae
DNA in CSFs revealed that 68% of CSF samples obtained from patients with MS were positive (11 out of 16 samples) for chlamydia DNA. Thus, the protocol established here is sensitive enough to detect chlamydia DNA from CSFs and can be used by other laboratories for evaluation of the presence of chlamydiae in CSFs because the protocol is based on the use of a commercial kit.
Borna Disease Virus and Human Disease
Authors: Carbone Kathryn M.
Published: July 2002

The biology of Borna disease virus (BDV) strongly supports the likelihood of human infection with BDV or a variant of BDV. Thus far, the evidence supporting BDV infection in humans has initiated much controversy among basic and clinical scientists; only time and additional research will support or refute the hypothesis of human BDV infection. Until an assay of acceptable specificity and sensitivity has been developed, validated, and used to document human BDV infection, scientists cannot reasonably begin to associate BDV infection with specific disease syndromes. Clinical studies seeking causal associations between BDV infection and specific diseases must ensure the proper identification of the BDV infection status of patients and control subjects by using a validated, highly sensitive, and highly specific assay (or series of assays). For clinical studies, a highly sensitive “screening” test followed by a highly specific confirmatory test will be of significant benefit. Although it is possible to formulate hypotheses about the clinical outcomes of human BDV infection based on animal model work, to date no human disease has been causally linked to human BDV infection. Scientists all over the world are actively pursuing these issues, and with continuing advances in clinical and basic BDV research, the answers cannot be far away.
Genetic Immunization of BALB/c mice with a Plasmid Bearing the Gene Coding for a Hybrid Merozoite Surface Protein 1-Hepatitis B Virus Surface Protein Fusion Protects Mice against LethalPlasmodium chabaudi chabaudiPC1 Infection
Authors: Wunderlich Gerhard , Moura Ivan C. , del Portillo Hernando A.
Published: July 2002

The genetic immunization of rodents with a plasmid coding for aPlasmodium chabaudimerozoite surface protein 1 (C terminus)-hepatitis B virus surface fusion protein (pPcMSP119-HBs) provided protection of mice against subsequent lethal challenge withP. chabaudi chabaudiPC1-infected red blood cells. The percentage of survivor mice was higher in DNA-immunized mice than in animals immunized with a recombinant rPcMSP119– glutathioneS-transferase fusion protein administered in Freund adjuvant. In all mice immunized with the pPcMSP119-HBs, a Th1-specific response, including the production of anti-MSP119-specific immunoglobulins predominantly of the immunoglobulin G2a subtype and reacting almost exclusively against discontinuous epitopes, was elicited. The coinjection of Th1-type cytokine-expressing plasmids (gamma interferon, interleukin-2, and granulocyte-macrophage colony-stimulating factor) mostly abolished protection and boosting of MSP119-specific antibodies. The inclusion of a lymph node-targeting signal did not significantly increase protection. These data provide further evidence that MSP119-HBs DNA constructs might be useful as components of a genetic vaccine against the asexual blood stages ofPlasmodium.
Formation of Methyl Mercaptan from
l
-Methionine by
Porphyromonas gingivalis
Authors: Yoshimura Mamiko , Nakano Yoshio , Yamashita Yoshihisa , Oho Takahiko , Saito Toshiyuki , Koga Toshihiko
Published: July 2002

Methyl mercaptan production by oral bacteria is thought to be one of the main causes of oral malodor. We examined the ability of periodontopathic
Porphyromonas gingivalis
to produce methyl mercaptan from
l
-methionine and found that the invasive strains W83 and W50 produced large amounts of methyl mercaptan. We cloned and sequenced the
mgl
gene encoding
l
-methionine-α-deamino-γ-mercaptomethane-lyase (METase) from
P. gingivalis
W83. The structural
mgl
gene consisted of 1,200 bp and encoded a 43.3-kDa protein. To examine the role of methyl mercaptan in the pathogenesis of
P. gingivalis
, a METase-deficient mutant of
P. gingivalis
W83 was constructed. The methionine degradation activity and virulence of the mutant (M1217) and the parent strain (W83) in mice were compared. M1217 showed a marked decrease in the formation of methyl mercaptan from
l
-methionine and decreased virulence compared with the wild-type strain W83. These results suggest that methyl mercaptan not only is one of the sources of oral malodor, but may also play a role in the pathogenicity of
P. gingivalis.
ESAT-6 Subunit Vaccination againstMycobacterium tuberculosis
Authors: Brandt Lise , Elhay Martin , Rosenkrands Ida , Lindblad Erik B. , Andersen Peter
Published: July 2002

The ESAT-6 antigen fromMycobacterium tuberculosisis a dominant target for cell-mediated immunity in the early phase of tuberculosis (TB) in TB patients as well as in various animal models. The purpose of our study was to evaluate the potential of ESAT-6 in an experimental TB vaccine. We started out using dimethyl dioctadecylammonium bromide (DDA), an adjuvant which has been demonstrated to be efficient for the induction of cellular immune responses and has been used successfully before as a delivery system for TB vaccines. Here we demonstrate that, whereas immune responses to both short-term-culture filtrate and Ag85B are efficiently induced with DDA, this adjuvant was inefficient for the induction of immune responses to ESAT-6. Therefore, we investigated the modulatory effect of monophosphoryl lipid A (MPL), an immunomodulator which in different combinations has demonstrated strong adjuvant activity for both cellular and humoral immune responses. We show in the present study that vaccination with ESAT-6 delivered in a combination of MPL and DDA elicited a strong ESAT-6-specific T-cell response and protective immunity comparable to that achieved withMycobacterium bovisBCG.
Shigella flexneri
2a Strain CVD 1207, with Specific Deletions in
virG
,
sen
,
set
, and
guaBA
, Is Highly Attenuated in Humans
Authors: Kotloff Karen L. , Noriega Fernando R. , Samandari Taraz , Sztein Marcelo B. , Losonsky Genevieve A. , Nataro James P. , Picking William D. , Barry Eileen M. , Levine Myron M.
Published: July 2002

A phase 1 clinical trial was conducted among 35 healthy adult volunteers to evaluate the safety, immunogenicity, and shedding of different doses of CVD 1207, a live attenuated
Shigella flexneri
2a vaccine candidate with specific deletion mutations in
virG
,
sen
,
set
, and
guaBA
. CVD 1207 retains the ability to invade epithelial cells but cannot effectively spread intercellularly after invasion (Δ
virG
), does not produce enterotoxin (Δ
sen
and Δ
set
), and has limited proliferation in vivo (Δ
guaBA
). In a consecutive fashion, groups of three to seven subjects ingested a single oral dose of CVD 1207 at an inoculum of either 10
6
, 10
7
, 10
8
, 10
9
, or 10
10
CFU. CVD 1207 was remarkably well-tolerated at inocula as high as 10
8
CFU. In comparison, one of 12 subjects who received 10
9
CFU experienced mild diarrhea and another experienced a single episode of emesis. One of five subjects who received 10
10
CFU experienced watery diarrhea and emesis. All subjects who ingested doses of 10
8
to 10
10
CFU excreted the vaccine; in 23 of 25, the duration of excretion was ≤3 days. A dose-related, immunoglobulin A antibody-secreting cell (ASC) response to
S. flexneri
2a O-specific lipopolysaccharide was seen, with geometric mean peak values of 6.1 to 35.2 ASCs/10
6
peripheral blood mononuclear cells (PBMC) among recipients of 10
7
to 10
10
CFU. The cytokine response to
Shigella
-specific antigens observed in volunteers' PBMC following vaccination suggested a Th1 pattern with stimulation of gamma interferon and absence of interleukin 4 (IL-4) or IL-5. CVD 1207 represents a
Shigella
live oral vaccine strain prepared from wild-type
S. flexneri
2a by rational use of recombinant DNA technology that achieves a remarkable degree of attenuation compared with earlier recombinant strains, even when administered at high dosage.
Cable-Piliated
Burkholderia cepacia
Binds to Cytokeratin 13 of Epithelial Cells
Authors: Sajjan Umadevi S. , Sylvester Francisco A. , Forstner Janet F.
Published: July 2002

Although the
Burkholderia cepacia
complex consists of several genomovars, one highly transmissible strain of
B. cepacia
has been isolated from the sputa of cystic fibrosis (CF) patients throughout the United Kingdom and Canada. This strain expresses surface cable (Cbl) pili and is thought to be the major strain associated with the fatal “cepacia syndrome.” In the present report we characterize the specific 55-kDa buccal epithelial cell (BEC) protein that binds cable pilus-positive
B. cepacia
. N-terminal sequences of CNBr-generated internal peptides identified the protein as cytokeratin 13 (CK13). Western blots of BEC extracts probed with a specific monoclonal antibody to CK13 confirmed the identification. Mixed epidermal cytokeratins (which contain CK13), cytokeratin extract from BEC (which consists essentially of CK13 and CK4), and a polyclonal antibody to mixed cytokeratins inhibited
B. cepacia
binding to CK13 blots and to normal human bronchial epithelial (NHBE) cells. Preabsorption of the antikeratin antibody with the BEC cytokeratin fraction reversed the inhibitory effect of the antibody. A cytokeratin mixture lacking CK13 was ineffective as an inhibitor of binding. Colocalization of CK13 and
B. cepacia
by confocal microscopy demonstrated that intact nonpermeabilized NHBE cells express small amounts of surface CK13 and bind Cbl-positive
B. cepacia
in the same location. Binding to intact NHBE cells was dependent on bacterial concentration and was saturable, whereas a Cbl-negative isolate exhibited negligible binding. These findings raise the possibility that surface-accessible CK13 in respiratory epithelia may be a biologically relevant target for the binding of cable piliated
B. cepacia
.
Effects of Estradiol and Progesterone on Susceptibility and Early Immune Responses toChlamydia trachomatisInfection in the Female Reproductive Tract
Authors: Kaushic Charu , Zhou Fan , Murdin Andrew D. , Wira Charles R.
Published: July 2002

We have used a previously described rodent model to examine the influence of hormonal environment on susceptibility and immune responses to genitalChlamydiainfection. Ovariectomized rats were administered estradiol, progesterone, or a combination of both, infected withChlamydia trachomatisvia the intrauterine route, and sacrificed 5 days later. Histopathological examination showed severe inflammation in the uteri and vaginae of progesterone-treated animals, whereas animals receiving estradiol or a combination of both hormones showed no inflammation. Large numbers of chlamydiae were found in vaginal secretions of progesterone-treated and combination-treated animals, while estradiol-treated animals had none. Tissue localization showed that numerous chlamydial inclusions were present in the uterine epithelium of the progesterone group and the cervicovaginal epithelium of the combination group. Examination of the acute immune responses of the infected animals showed that maximum activation was present in the draining lymph node cells from the progesterone-treated group, and these cells were producing large amounts of interleukin-10 and gamma interferon compared to other hormone-treated groups. In contrast, spleen cell proliferation was suppressed in progesterone-treated animals compared to other hormone-treated groups. We conclude that progesterone increases and estradiol decreases susceptibility to intrauterine chlamydial infection in this rat model. Our data demonstrate that hormone environment, at the time of infection, has a profound effect on the outcome of microbial infection in the female reproductive tract.
β-1,2-Linked Oligomannosides fromCandida albicansBind to a 32-Kilodalton Macrophage Membrane Protein Homologous to the Mammalian Lectin Galectin-3
Authors: Fradin Chantal , Poulain Daniel , Jouault Thierry
Published: July 2002

β-1,2-linked oligomannoside residues are present, associated with mannan and a glycolipid, the phospholipomannan, at theCandida albicanscell wall surface. β-1,2-linked oligomannoside residues act as adhesins for macrophages and stimulate these cells to undergo cytokine production. To characterize the macrophage receptor involved in the recognition ofC. albicansβ-1,2-oligomannoside we used the J774 mouse cell line, which is devoid of the receptor specific for α-linked mannose residues. A series of experiments based on affinity binding on eitherC. albicansyeast cells or β-1,2-oligomannoside-conjugated bovine serum albumin (BSA) and subsequent disclosure with biotinylated conjugated BSA repeatedly led to the detection of a 32-kDa macrophage protein. An antiserum specific for this 32-kDa protein inhibitedC. albicansbinding to macrophages and was used to immunoprecipitate the molecule. Two high-pressure liquid chromatography-purified peptides from the 32-kDa tryptic digest showed complete homology to galectin-3 (previously designated Mac-2 antigen), an endogenous lectin with pleiotropic functions which is expressed in a wide variety of cell types with whichC. albicansinteracts as a saprophyte or a parasite.
Protection against
Streptococcus pneumoniae
Elicited by Immunization with Pneumolysin and CbpA
Authors: Ogunniyi Abiodun David , Woodrow Matthew C. , Poolman Jan T. , Paton James C.
Published: July 2002

The need for the development of cheap and effective vaccines against pneumococcal disease has necessitated the evaluation of common virulence-associated proteins of
Streptococcus pneumoniae
as potential vaccine antigens. In this study, we examined the capacity of active immunization with a genetic toxoid derivative of pneumolysin (PdB) and/or a fragment of choline binding protein A (CbpA; also known as PspC, Hic, and SpsA) to protect mice from intraperitoneal challenge with medium to very high doses of a highly virulent capsular type 2 pneumococcal strain, D39. The median survival times for mice immunized with the individual protein antigens in different adjuvant combinations were significantly longer than those for mice that received the respective adjuvants alone. Mice immunized with CbpA alone were significantly better protected than mice immunized with PdB alone. Correspondingly, the median survival times for mice that were immunized with a combination of PdB and CbpA were significantly longer than those for mice that received PdB alone but not significantly different from those that received CbpA alone. Mice immunized with the protein antigens in a mixture of monophospholipid A (MPL) and aluminium phosphate (AlPO
4
) adjuvants had higher antibody titers than mice that received the antigens in AlPO
4
alone. Mice immunized with PdB in MPL plus AlPO
4
were also significantly better protected than mice that received PdB in AlPO
4
alone.
Cryptosporidium parvum-Specific Mucosal Immune Response in C57BL/6 Neonatal and Gamma Interferon-Deficient Mice: Role of Tumor Necrosis Factor Alpha in Protection
Authors: Lacroix Sonia , Mancassola Roselyne , Naciri Muriel , Laurent Fabrice
Published: July 2002

Both neonatal and C57BL/6 gamma interferon (IFN-γ) knockout (C57BL/6-GKO) mice are susceptible toCryptosporidium parvum, but the course of infection is different. Neonatal mice are able to clear the parasite within 3 weeks, whereas C57BL/6-GKO mice, depending on age, die rapidly or remain chronically infected. The mechanism by which IFN-γ leads to a protective immunity is yet poorly understood. In order to investigate the effect of IFN-γ on other cytokines expressed in the intestinal mucosa duringC. parvuminfection, we studied cytokine mRNA expression in the neonates and GKO (neonatal and adult) mice by quantitative reverse transcription-PCR (RT-PCR) at 4 and 9 days after infection. IFN-γ mRNA levels were quickly and strongly up-regulated in the mucosa of neonatal mice. In GKO mice, the Th1-type response was dramatically altered during the infection, whereas the mRNA expression levels of the Th2-type cytokines interleukin 4 (IL-4) and IL-10 were increased in both mouse models. In the absence of IFN-γ, the adult knockout mice up-regulated the mRNA levels of inflammatory cytokines, such as IL-1β, IL-6, and granulocyte-macrophage colony-stimulating factor, in the mucosa, but not tumor necrosis factor alpha (TNF-α), whereas all these cytokines were up-regulated in the infected neonatal mice. Further experiments indicated that injections of TNF-α into GKO adult mice significantly reduced oocyst shedding. The results of the present study indicate that the resolution of infection is dependent on the expression of Th1-type cytokines in the mucosa of C57BL/6 mice and that TNF-α may participate in the control of parasite development.
Rhoptry-Associated Protein 1-Binding Monoclonal Antibody Raised against a Heterologous Peptide Sequence InhibitsPlasmodium falciparumGrowth In Vitro
Authors: Moreno Rafael , Pöltl-Frank Friederike , Stüber Dietrich , Matile Hugues , Mutz Michael , Weiss Niklaus A. , Pluschke Gerd
Published: July 2002

Monoclonal antibodies (MAbs) specific forPlasmodium falciparumrhoptry-associated protein 1 (RAP-1) were generated and tested for inhibition of parasite growth in vitro. The majority of indirect immunofluorescence assay (IFA)-positive MAbs raised against recombinant RAP-1 positions 23 to 711 (rRAP-123–711) recognized epitopes located in the immunodominant N-terminal third of RAP-1. MAbs specific for the building block 35.1 of the synthetic peptide malaria vaccine SPf66 also yielded an IFA staining pattern characteristic for rhoptry-associated proteins and reacted specifically with rRAP-1 and parasite-derived RAP-1 molecules p67 and p82. Cross-reactivity with RAP-1 was blocked by the 35.1 peptide. Epitope mapping with truncated rRAP-1 molecules and overlapping peptides identified the linear RAP-1 sequence Y218KYSL222as a target of the anti-35.1 MAbs. This sequence lacks primary sequence similarity with the 35.1 peptide (YGGPANKKNAG). Cross-reactivity of the anti-35.1 MAbs thus appears to be associated with conformational rather than sequence homology. While the anti-35.1 MAb SP8.18 exhibited parasite growth-inhibitory activity, none of the tested anti-rRAP-123–711MAbs inhibited parasite growth, independently of their fine specificity for the RAP-1 sequences at positions 33 to 42, 213 to 222, 243 to 247, 280 to 287, or 405 to 446. The growth-inhibitory activity of MAb SP8.18 was, however, accelerated by noninhibitory anti-RAP-1 MAbs. Results demonstrate that in addition to fine specificity, other binding parameters are also crucial for the inhibitory potential of an antibody.
Boosting Vaccine for Tuberculosis
Authors: Brooks Jason V. , Frank Anthony A. , Keen Marc A. , Bellisle John T. , Orme Ian M.
Published: July 2002

An effective new vaccine for the control of tuberculosis is badly needed. While current research attempts to improve vaccination are concentrating on new prophylactic or immunotherapeutic vaccines, virtually no emphasis has been placed on boosting individuals already inoculated with
Mycobacterium bovis
BCG. It is shown here that mice vaccinated with BCG gradually lose their capacity to resist an aerosol challenge infection as they age. However, if these mice are inoculated with the 30-kDa mycolyl transferase A protein in midlife, after challenge when aged they express levels of protection in the lungs comparable to those of young mice, associated with minimal pathological damage.
Intranasal Immunization Enhances Clearance of NontypeableHaemophilus influenzaeand Reduces Stimulation of Tumor Necrosis Factor Alpha Production in the Murine Model of Otitis Media
Authors: Sabirov Albert , Kodama Satoru , Hirano Takashi , Suzuki Masashi , Mogi Goro
Published: July 2002

NontypeableHaemophilus influenzae(NTHi) is a major pathogen causing otitis media (OM). One of the outer membrane proteins of NTHi, P6, is a common antigen to all strains and is considered a candidate for mucosal vaccine. We have previously reported that intranasal immunization with P6 and cholera toxin (CT) could induce P6-specific immunoglobulin A (IgA) antibodies in the middle ear. In the present study, we assessed the effect of intranasal immunization for the protection against NTHi-induced OM. Mice were immunized intranasally with P6 and CT as an adjuvant on days 0, 7, and 14. Control mice were given phosphate-buffered saline (PBS) without antigen. One week after the final immunization, a suspension of live NTHi (107CFU) was injected into the tympanic cavity to induce experimental OM. On days 3 and 7 after bacterial challenge, mice were killed and middle ear effusions (MEEs) were collected. All immunized mice showed elevated titers of P6-specific antibodies in MEEs. The rank order of specific antibody included, from highest to lowest levels, IgG, IgA, and IgM. In addition, immunized mice showed enhanced clearance of NTHi from the middle ear and the number of NTHi in MEEs of immunized mice was reduced by 97% on day 3 and by 92% on day 7 after bacterial challenge relative the number in the MEEs of control mice. The protective effect of intranasal immunization on the incidence of NTHi-induced experimental OM was evident on day 7 after challenge. By day 7, the number of MEEs in immunized mice was 64% less than that in control mice and the incidence of NTHi culture-positive MEEs in immunized mice was 56% less than that in control mice. Less stimulation of tumor necrosis factor alpha (TNF-α) production in the middle ear was evident on day 3 after challenge. Immunized mice showed lower concentrations of TNF-α in MEEs. These results indicate that intranasal immunization affords protection against experimental OM as evidenced by enhanced clearance of NTHi and less stimulation of TNF-α production in the middle ear. These findings suggest that a nasal vaccine might be useful for preventing OM.
Polysaccharide Biosynthesis Locus Required for Virulence ofBacteroides fragilis
Authors: Coyne Michael J. , Tzianabos Arthur O. , Mallory Benjamin C. , Carey Vincent J. , Kasper Dennis L. , Comstock Laurie E.
Published: July 2002

Bacteroides fragilis,though only a minor component of the human intestinal commensal flora, is the anaerobe most frequently isolated from intra-abdominal abscesses.B. fragilis9343 expresses at least three capsular polysaccharides—polysaccharide A (PS A), PS B, and PS C. Purified PS A and PS B have been tested in animal models and are both able to induce the formation of intra-abdominal abscesses. Mutants unable to synthesize PS B or PS C still facilitate abscess formation at levels comparable to those of wild-type 9343. To determine the contribution of PS A to abscess formation in the context of the intact organism, the PS A biosynthesis region was cloned, sequenced, and deleted from 9343 to produce a PS A-negative mutant. Animal experiments demonstrate that the abscess-inducing capability of 9343 is severely attenuated when the organism cannot synthesize PS A, despite continued synthesis of the other capsular polysaccharides. The PS A of 9343 contains an unusual free amino sugar that is essential for abscess formation by this polymer. PCR analysis of the PS A biosynthesis loci of 50B. fragilisisolates indicates that regions flanking each side of this locus are conserved in all strains. The downstream conserved region includes two terminal PS A biosynthesis genes that homology-based analyses predict are involved in the synthesis and transfer of the free amino sugar of PS A. Conservation of these genes suggests that this sugar is present in the PS A of all serotypes and may explain the abscessogenic nature ofB. fragilis.
Neutrophil Depletion duringToxoplasma gondiiInfection Leads to Impaired Immunity and Lethal Systemic Pathology
Authors: Bliss Susan K. , Gavrilescu L. Cristina , Alcaraz Ana , Denkers Eric Y.
Published: July 2002

The immunomodulatory role of neutrophils during infection withToxoplasma gondiiwas investigated. Monoclonal antibody-mediated depletion revealed that neutrophils are essential for survival during the first few days of infection. Moreover, neutrophil depletion was associated with a weaker type 1 immune response as measured by decreased levels of gamma interferon, interleukin-12 (IL-12) and tumor necrosis factor alpha. IL-10 was also decreased in depleted animals. Additionally, splenic populations of CD4+T cells, CD8+T cells, and NK1.1+cells were decreased in depleted mice. Neutrophil-depleted mice exhibited lesions of greater severity in tissues examined and a greater parasite burden as determined by histopathology and reverse transcription-PCR. We conclude that neutrophils are critical near the time of infection because they influence the character of the immune response and control tachyzoite replication.
Role of Trehalose Dimycolate in Recruitment of Cells and Modulation of Production of Cytokines and NO in Tuberculosis
Authors: Lima Valéria M. F. , Bonato Vania L. D. , Lima Karla M. , Dos Santos Sandra A. , Dos Santos Rubens R. , Gonçalves Eduardo D. C. , Faccioli Lucia H. , Brandão Izaira T. , Rodrigues-Junior José M. , Silva Célio L.
Published: July 2002

Mice treated with viableMycobacterium tuberculosiswith no glycolipid trehalose dimycolate (TDM) on the outer cell wall (delipidatedM. tuberculosis) by intraperitoneal or intratracheal inoculation presented an intense recruitment of polymorphonuclear cells into the peritoneal cavity and an acute inflammatory reaction in the lungs, respectively. In addition, lung lesions were resolved around the 32nd day after intratracheal inoculation. TDM-loaded biodegradable poly-dl-lactide-coglycolide microspheres as well as TDM-coated charcoal particles induced an intense inflammatory reaction. In addition, high levels of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), IL-12, IL-10, gamma interferon (IFN-γ), and IL-4 production were detected in lung cells, and nitric oxide (NO) production was high in culture supernatants of bronchoalveolar lavage cells. These in vivo data were confirmed by in vitro experiments using peritoneal macrophages cultured in the presence of TDM adsorbed onto coverslips. High levels of IFN-γ, IL-6, TNF-α, IL-12, IL-10, and NO were detected in the culture supernatants. Our results suggest that TDM contributes to persistence of infection through production of cytokines, which are important for the recruitment of inflammatory cells and maintenance of a granulomatous reaction. In addition, our findings are important for a better understanding of the immunostimulatory activity of TDM and its possible use as an adjuvant in experiments using DNA vaccine or gene therapy against tuberculosis.
Changes in Availability of Oxygen Accentuate Differences in Capsular Polysaccharide Expression by Phenotypic Variants and Clinical Isolates of
Streptococcus pneumoniae
Authors: Weiser Jeffrey N. , Bae Deborah , Epino Henry , Gordon Stephen B. , Kapoor Miki , Zenewicz Lauren A. , Shchepetov Mikhail
Published: July 2002

Most isolates of
Streptococcus pneumoniae
are mixed populations of transparent (T) and opaque (O) colony phenotypes. Differences in the production of capsular polysaccharide (CPS) between O and T variants were accentuated by changes in the environmental concentration of oxygen. O variants demonstrated a 5.2- to 10.6-fold increase in amounts of CPS under anaerobic compared to atmospheric growth conditions, while CPS production remained low under all conditions for T variants. Increased amounts of CPS in O compared to T pneumococci were associated with increased expression of
cps
-encoded proteins. The inhibitory effect of oxygen on expression of CPS in O variants correlated with decreased tyrosine phosphorylation of CpsD, a tyrosine kinase and regulator of CPS synthesis. Modulation of CpsD expression and its activity by tyrosine phosphorylation may allow the pneumococcus to adapt to the requirements of both colonization, where decreased CPS allows for adherence, and bacteremia, where increased CPS may be required to escape from opsonic clearance. In patients with invasive infection, paired isolates from the same patient were shown to have predominately a T colony phenotype without phosphotyrosine on CpsD when cultured from the nasopharynx, and an O phenotype that phosphorylates CpsD in response to oxygen when cultured from the blood. Differences in the availability of oxygen, therefore, may be a key factor in allowing for the selection of distinct phenotypes in these two host environments.
Laccase of
Cryptococcus neoformans
Is a Cell Wall-Associated Virulence Factor
Authors: Zhu Xudong , Gibbons Jack , Garcia-Rivera Javier , Casadevall Arturo , Williamson Peter R.
Published: July 2002

Virulence is the outcome of an interaction between the host and a microbe and is characterized by a large array of opposing reactions operating at the host-pathogen interface.
Cryptococcus neoformans
is an important opportunistic pathogen in immunocompromised patients, including those with human immunodeficiency virus, and expresses a virulence-associated laccase which is believed to oxidize brain catecholamines and iron as a defense against host immune cells. In the present report, we investigated the cellular location of laccase to understand more fully how it contributes to cryptococcal virulence. A monoclonal antibody to the
C. neoformans
laccase was generated and used to show localization in the cell walls of representative serotype A (H99) and serotype D (B-3501) strains by immunoelectron microscopy. In addition, confocal microscopy was used to show a peripheral location of green fluorescent protein-tagged laccase expressed in live H99 cells. Biochemical studies showed that laccase could be released from intact cells or cell wall fractions with glucanase enzymes but was retained in the cell wall after sequential extraction with 1 M NaCl, 6 M urea, and 1% sodium dodecyl sulfate. The presence of a hydrolyzable bond linking laccase to the cell wall was suggested by removal of laccase from cell wall preparations after they were boiled in 1% sodium dodecyl sulfate, as was the presence of a disulfide or thioester bond by removal with dithiothreitol or β-mercaptoethanol. These data show that laccase is present as a tightly associated cell wall enzyme that is readily accessible for interactions with host immune cells.
Immunodominant Epitopes inBabesia bovisRhoptry-Associated Protein 1 That Elicit Memory CD4+-T-Lymphocyte Responses inB. bovis-Immune Individuals Are Located in the Amino-Terminal Domain
Authors: Norimine Junzo , Suarez Carlos E. , McElwain Terry F. , Florin-Christensen Monica , Brown Wendy C.
Published: July 2002

Babesia bovisrhoptry-associated protein 1 (RAP-1), which confers partial protection againstB. bovischallenge, is recognized by antibodies and T lymphocytes from cattle that have recovered from infection and are immune to subsequent challenge. RAP-1 is a 60-kDa protein with an N-terminal (NT) region that contains four cysteine residues conserved among allBabesiaRAP-1 family members and a C-terminal (CT) region that contains multiple, degenerate, tandem 23-amino-acid (aa) repeats. To define the location of CD4+-T-cell epitopes for vaccine development using a recombinant protein or minigene construct, a series of truncated recombinant RAP-1 proteins and peptides were tested for stimulation of T-cell lines derived fromB. bovis-immune cattle. CD4+-T-cell lines from threeB. bovis-immune cattle with differentDRB3haplotypes responded to the NT region of RAP-1, whereas T cells from only one animal responded weakly to the CT region. T-cell lines from the three individuals recognized two to six NT-region peptides spanning aa 134 to 316 and representing at least four dominant epitopes. Using RAP-1-specific CD4+-T-cell clones, two NT-region epitopes, EYLVNKVLYMATMNYKT (aa 187 to 203) and EAPWYKRWIKKFR (aa 295 to 307), and one CT-region repeat epitope, FREAPQATKHFL, which is present twice at aa positions 391 to 402 and 414 to 425, were identified. Several peptides representing degenerate repeats of the agonist CT-region peptide FREAPQATKHFL neither stimulated responses of T-cell clones specific for this peptide nor inhibited responses to the agonist peptide. Upon stimulation with specific antigen, T-cell clones specific for NT or CT epitopes produced gamma interferon. The presence of T-helper-cell epitopes in the NT domain of RAP-1, which is highly conserved among otherwise antigenically different strains ofB. bovis, supports the inclusion of this region in vaccine constructs to be tested in cattle.
The In Vitro Interaction ofStreptococcus pyogeneswith Human Pharyngeal Cells Induces a Phage-Encoded Extracellular DNase
Authors: Broudy Thomas B. , Pancholi Vijaykumar , Fischetti Vincent A.
Published: July 2002

The role lysogenic bacteriophage play in the pathogenesis of the host bacterium is poorly understood. In a previous study, we found that streptococcal coculture with human pharyngeal cells resulted in the induction of lysogenic bacteriophage as well as the phage-associated streptococcal pyrogenic exotoxin C (SpeC). In this study, we have determined that in addition to SpeC induction, a number of other streptococcal proteins are also released by the bacteria during coculture with pharyngeal cells. Among these, we identified and characterized a novel 27-kDa secreted protein. Sequence analysis of this novel protein demonstrated it to be encoded by the same lysogenic bacteriophage which harborsspeC. Protein sequence analysis revealed varied homologies with several streptococcal DNases. Further biochemical characterization of the recombinantly expressed protein verified it to be a divalent cation-dependent streptococcal phage-encoded DNase (Spd1). Although functionally distinct, SpeC and Spd1 are associated by a number of parameters, including genetic proximity and transcriptional regulation. Finally, we speculate on the induction of phage-encoded DNase (Spd1) enhancing the fitness of both bacteria and phage.
Entamoeba histolytica
Lectins Contain Unique 6-Cys or 8-Cys Chitin-Binding Domains
Authors: Van Dellen Katrina , Ghosh Sudip K. , Robbins Phillips W. , Loftus Brendan , Samuelson John
Published: July 2002

The Jacob lectin, the most abundant glycoprotein in the cyst wall of
Entamoeba invadens
, contains five unique 6-Cys chitin-binding domains (CBDs). We identified
Entamoeba histolytica
and
Entamoeba dispar
genes encoding Jacob homologues, each of which contains two predicted 6-Cys CBDs. A unique 8-Cys CBD was found at the N termini of the
E. histolytica
chitinase and three other predicted lectins, called Jessie 1 to Jessie 3. The CBDs of four
E. histolytica
lectins (Jacob, chitinase, and Jessies 2 and 3) were expressed in secretory vesicles of transfected amebae and shown to bind to particulate chitin.
Dual Control of Streptokinase and Streptolysin S Production by thecovRSandfasCAXTwo-Component Regulators inStreptococcus dysgalactiaesubsp.equisimilis
Authors: Steiner Kerstin , Malke Horst
Published: July 2002

Synthesis of the plasminogen activator streptokinase (SK) by group A streptococci (GAS) has recently been shown to be subject to control by two two-component regulators,covRS(orcsrRS) andfasBCA. In independent studies, response regulator CovR proved to act as the repressor, whereas FasA was found to act indirectly as the activator by controlling the expression of a stimulatory RNA,fasX. In an attempt at understanding the regulation of SK production in the human group C streptococcal (GCS) strain H46A, the strongest SK producer known yet, we provide here physical and functional evidence for the presence of thecovandfassystems in GCS as well and, using a mutational approach, compare the balance between their opposing actions in H46A and GAS strain NZ131. Sequence analysis combined with Southern hybridization revealed that thecovRSandfasCAXoperons are preserved at high levels of primary structure identity between the corresponding GAS and GCS genes, with the exception offasB, encoding a second sensor kinase that is not a member of the GCSfasoperon. This analysis also showed that wild-type H46A is actually a derepressed mutant for SK and streptolysin S (SLS) synthesis, carrying a K102 amber mutation incovR.Usingcovandfasmutations in various combinations together with strain constructs allowing complementation intrans, we found that, in H46A,covandfascontribute to approximately equal negative and positive extents, respectively, to constitutive SK and SLS activity. The amounts of SK paralleled the level ofskcH46Atranscription. The most profound difference between H46A and NZ131 regarding the relative activities of thecovandfassystems consisted in significantly higher activity of a functional CovR repressor in NZ131 than in H46A. In NZ131, CovR decreased SK activity in a Fas+background about sevenfold, compared to a 1.9-fold reduction of SK activity in H46A. Combined with the very short-lived nature ofcovRmRNA (decay rate, 1.39/min), such differences may contribute to strain-specific peculiarities of the expression of two prominent streptococcal virulence factors in response to environmental changes.
Chronic Helminth Infection Induces Alternatively Activated Macrophages Expressing High Levels of CCR5 with Low Interleukin-12 Production and Th2-Biasing Ability
Authors: Rodríguez-Sosa Miriam , Satoskar Abhay R. , Calderón Rodrigo , Gomez-Garcia Lorena , Saavedra Rafael , Bojalil Rafael , Terrazas Luis I.
Published: July 2002

Helminth infections induce Th2-type biased immune responses. Although the mechanisms involved in this phenomenon are not yet clearly defined, antigen-presenting cells (APC) could play an important role in this process. Here, we have used peritoneal macrophages (F4/80+) recruited at different times after challenge withTaenia crassicepsas APC and tested their ability to regulate Th1/Th2 differentiation. Macrophages from acute infections produced high levels of interleukin-12 (IL-12) and nitric oxide (NO), paralleled with low levels of IL-6 and prostaglandin E2(PGE2) and with the ability to induce strong antigen-specific CD4+ T-cell proliferation in response to nonrelated antigens. In contrast, macrophages from chronic infections produced higher levels of IL-6 and PGE2and had suppressed production of IL-12 and NO, associated with a poor ability to induce antigen-specific proliferation in CD4+ T cells. Failure to induce proliferation was not due to a deficient expression of accessory molecules, since major histocompatibility complex class II, CD40, and B7-2 were up-regulated, together with CD23 and CCR5 as infection progressed. These macrophages from chronic infections were able to bias CD4+ T cells to produce IL-4 but not gamma interferon (IFN-γ), contrary to macrophages from acute infections. Blockade of B7-2 and IL-6 and inhibition of PGE2failed to restore the proliferative response in CD4+ T cells. Furthermore, studies using STAT6−/−mice revealed that STAT6-mediated signaling was essential for the expansion of these alternatively activated macrophages. These data demonstrate that helminth infections can induce different macrophage populations that have Th2-biasing properties.
Strain-Dependent Induction of Enterocyte Apoptosis byGiardia lambliaDisrupts Epithelial Barrier Function in a Caspase-3-Dependent Manner
Authors: Chin Alex C. , Teoh Desiree A. , Scott Kevin G.-E. , Meddings Jonathon B. , Macnaughton Wallace K. , Buret Andre G.
Published: July 2002

We recently demonstrated thatGiardia lambliarearranges cytoskeletal proteins and reduces transepithelial electrical resistance. The effect ofG. lambliaon enterocyte apoptosis is unknown, and a possible link between microbially induced enterocyte apoptosis and altered epithelial permeability has yet to be established. The aim of this study was to assess whetherG. lambliainduces enterocyte apoptosis in duodenal epithelial monolayers and whether this effect increases epithelial permeability. Monolayers of nontransformed human duodenal epithelial cells were incubated with sonicated or liveG. lambliatrophozoites (NF, S2, WB, or PB strains) for 8, 24, and 48 h. Cell cultures were assessed for apoptosis by Hoechst fluorescence staining, enzyme-linked immunosorbent assay for apoptotic nucleosomes, and electron microscopy. In separate experiments, monolayers were pretreated with or without 120 μM caspase-3 inhibitor (Z-DEVD-FMK) for 1 h and were assessed for production of apoptotic nucleosomes, tight junctional integrity (with fluorescent ZO-1 staining followed by confocal laser microscopy), and transepithelial permeability for fluorescein isothiocyanate-dextran.G. lambliastrains NF and S2, but not strains WB or PB, induced enterocyte apoptosis within the monolayers, and this effect was inhibited by Z-DEVD-FMK pretreatment. Using theG. lambliaNF isolate, additional experiments investigated the possible link between enterocyte apoptosis and altered epithelial permeability.G. lambliaNF disrupted tight junctional ZO-1 and increased epithelial permeability, but these effects were also prevented by pretreatment with the caspase-3 inhibitor. These findings indicate that strain-dependent induction of enterocyte apoptosis may contribute to the pathogenesis of giardiasis. This effect is responsible for a loss of epithelial barrier function by disrupting tight junctional ZO-1 and increasing permeability in a caspase-3-dependent manner.
Catabolism of α-Ketoglutarate by a
sucA
Mutant of
Bradyrhizobium japonicum
: Evidence for an Alternative Tricarboxylic Acid Cycle
Authors: Green Laura S. , Li Youzhong , Emerich David W. , Bergersen Fraser J. , Day David A.
Published: July 2002

A complete tricarboxylic acid (TCA) cycle is generally considered necessary for energy production from the dicarboxylic acid substrates malate, succinate, and fumarate. However, a
Bradyrhizobium japonicum sucA
mutant that is missing α-ketoglutarate dehydrogenase is able to grow on malate as its sole source of carbon. This mutant also fixes nitrogen in symbiosis with soybean, where dicarboxylic acids are its principal carbon substrate. Using a flow chamber system to make direct measurements of oxygen consumption and ammonium excretion, we confirmed that bacteroids formed by the
sucA
mutant displayed wild-type rates of respiration and nitrogen fixation. Despite the absence of α-ketoglutarate dehydrogenase activity, whole cells of the mutant were able to decarboxylate α-[U-
14
C]ketoglutarate and [U-
14
C]glutamate at rates similar to those of wild-type
B. japonicum
, indicating that there was an alternative route for α-ketoglutarate catabolism. Because cell extracts from
B. japonicum
decarboxylated [U-
14
C]glutamate very slowly, the γ-aminobutyrate shunt is unlikely to be the pathway responsible for α-ketoglutarate catabolism in the mutant. In contrast, cell extracts from both the wild type and mutant showed a coenzyme A (CoA)-independent α-ketoglutarate decarboxylation activity. This activity was independent of pyridine nucleotides and was stimulated by thiamine PP
i
. Thin-layer chromatography showed that the product of α-ketoglutarate decarboxylation was succinic semialdehyde. The CoA-independent α-ketoglutarate decarboxylase, along with succinate semialdehyde dehydrogenase, may form an alternative pathway for α-ketoglutarate catabolism, and this pathway may enhance TCA cycle function during symbiotic nitrogen fixation.
Relative Roles of the
fla/che
P
A
, P
D-3
, and P

sigD

Promoters in Regulating Motility and
sigD
Expression in
Bacillus subtilis
Authors: West Joyce T. , Estacio William , Márquez-Magaña Leticia
Published: July 2002

Three promoters have been identified as having potentially important regulatory roles in governing expression of the
fla/che
operon and of
sigD
, a gene that lies near the 3′ end of the operon. Two of these promoters,
fla/che
P
A
and P
D-3
, lie upstream of the >26-kb
fla/che
operon. The third promoter, P

sigD

, lies within the operon, immediately upstream of
sigD. fla/che
P
A
, transcribed by Eς
A
, lies ≥24 kb upstream of
sigD
and appears to be largely responsible for
sigD
expression. P
D-3
, transcribed by Eς
D
, has been proposed to participate in an autoregulatory positive feedback loop. P

sigD

, a minor ς
A
-dependent promoter, has been implicated as essential for normal expression of the
fla/che
operon. We tested the proposed functions of these promoters in experiments that utilized strains that bear chromosomal deletions of
fla/che
P
A
, P
D-3
, or P

sigD

. Our analysis of these strains indicates that
fla/che
P
A
is absolutely essential for motility, that P
D-3
does not function in positive feedback regulation of
sigD
expression, and that P

sigD

is not essential for normal
fla/che
expression. Further, our results suggest that an additional promoter(s) contributes to
sigD
expression.