bluuebunny/crossref_metadata_2025 huggingface dataset:
Activity of BMS-284756, a Novel Des-Fluoro(6) Quinolone, against
Staphylococcus aureus
, Including Contributions of Mutations to Quinolone Resistance
Authors: Low D. E. , Muller M. , Duncan C. L. , Willey B. M. , de Azavedo J. C. , McGeer A. , Kreiswirth B. N. , Pong-Porter S. , Bast D. J.
Published: July 2002

The in vitro activity of BMS-284756 against 602
Staphylococcus aureus
isolates, including 152 that were both methicillin and ciprofloxacin resistant (MIC ≥4 μg/ml), was determined. For ciprofloxacin-susceptible and nonsusceptible isolates, the MICs at which 50% of organisms were inhibited were 0.015 and 2 μg/ml and the MICs at which 90% of organisms were inhibited were 0.03 and 4 μg/ml, respectively.
Determining Linezolid’s Baseline In Vitro Activity in Canada Using Gram-Positive Clinical Isolates Collected prior to Its National Release
Authors: Karlowsky James A. , Kelly Laurie J. , Critchley Ian A. , Jones Mark E. , Thornsberry Clyde , Sahm Daniel F.
Published: July 2002

All of the isolates of
Staphylococcus aureus
(
n
= 317),
Enterococcus
species (
n
= 315),
Streptococcus pneumoniae
(
n
= 282), and
Staphylococcus epidermidis
(
n
= 176) collected at 16 Canadian microbiology laboratories from October 2000 to April 2001 were susceptible to linezolid. Future studies will determine how linezolid clinical use in Canada affects its in vitro activity.
Observation of Soft Contact Lens Disinfection with Fluorescent Metabolic Stains
Authors: Gavin J. , Button N. F. , Watson-Craik I. A. , Logan N. A.
Published: July 2002

A rapid fluorescent staining method using a tetrazolium dye and propidium iodide for the in-use assessment of disinfection of
Pseudomonas aeruginosa
biofilms on soft contact lenses showed that 11 to 13% of cells on lenses remained actively respiring and recoverable by culture methods after 30 min of exposure to 3% hydrogen peroxide.
Immunomagnetic Purification of
Colletotrichum lindemuthianum
Appressoria
Authors: Hutchison Katie A. , Perfect Sarah E. , O'Connell Richard J. , Green Jonathan R.
Published: July 2002

We developed a method to purify appressoria of the bean anthracnose fungus
Colletotrichum lindemuthianum
for biochemical analysis of the cell surface and to compare appressoria with other fungal structures. We used immunomagnetic separation after incubation of infected bean leaf homogenates with a monoclonal antibody that binds strongly to the appressoria. Preparations with a purity of >90% could be obtained. Examination of the purified appressoria by transmission electron microscopy showed that most had lost their cytoplasm. However, the plasma membrane was retained, suggesting that there is some form of attachment of this membrane to the cell wall. The purified appressoria can be used for studies of their cell surface, and we have shown that there are clear differences in the glycoprotein constituents of cell walls of appressoria compared with mycelium.
New Method for Evaluation of Virucidal Activity of Antiseptics and Disinfectants
Authors: Papageorgiou Georgios T. , Mocé-Llivina Laura , Jofre Juan
Published: July 2002

Counting culturable viruses adsorbed to cellulose nitrate filters (the VIRADEN method) is proposed as a simple procedure for the evaluation of the virucidal activity of antiseptics and disinfectants. The virucidal activities of two different doses of iodine, chlorine, glutaraldehyde, and chlorhexidine digluconate on poliovirus 1 were tested with a standardized procedure and with the VIRADEN method. The two procedures assayed provided similar results.
Cultivation of Bacteria Producing Polyamino Acids with Liquid Manure as Carbon and Nitrogen Source
Authors: Pötter Markus , Oppermann-Sanio Fred Bernd , Steinbüchel Alexander
Published: July 2002

Poly(γ-
d
-glutamic acid) (PGA)-producing strains of
Bacillus
species were investigated to determine their ability to contribute to reducing the amount of ammonium nitrogen in liquid manures and their ability to convert some of the ammonium into this polyamino acid as a transient depot for nitrogen. Organisms that do these things should help solve the serious environmental problems which are caused by the use of large amounts of liquid manure resulting from intensified agriculture; these problems are mainly due to the high content of ammonium nitrogen.
Bacillus licheniformis
ATCC 9945 and
Bacillus subtilis
were able to grow in liquid manure and to produce PGA in the presence of sodium gluconate. On artificial liquid manure these two strains were able to produce 0.85 and 0.79 g of PGA per liter, respectively. Under conditions that are found in intensified farming situations the ammonia content was reduced within 48 h from 1.3 to 0.75 g/liter. One mutant of
B. subtilis
1551 impaired in the catabolism of PGA was obtained after nitrosoguanidine mutagenesis. This mutant produced PGA at a final concentration of 4.8 g/liter, whereas the wild type produced only 3.7 g/liter.
Biological Activities and Structural Properties of the Atypical Bacteriocins Mesenterocin 52B and Leucocin B-TA33a
Authors: Corbier C. , Krier F. , Mulliert G. , Vitoux B. , Revol-Junelles A.-M.
Published: July 2002

The antibacterial spectra and modes of action of synthetic peptides corresponding to mesenterocin 52B and leucocin B-TA33a greatly differ despite their high sequence homology. Circular dichroism experiments establish the capacity of each of these two peptides to partly fold into an amphiphilic helix that might be crucial for their adsorption at lipophilic-hydrophilic interfaces.
Fiber-Optic Fluorometer for Microscale Mapping of Photosynthetic Pigments in Microbial Communities
Authors: Thar Roland , Kühl Michael , Holst Gerhard
Published: July 2002

Microscale fluorescence measurements were performed in photosynthetic biofilms at a spatial resolution of 100 to 200 μm with a new fiber-optic fluorometer which allowed four different excitation and emission wavelengths and was configured for measuring phycobiliproteins, chlorophylls, and bacteriochlorophylls. We present details of the measuring system and describe examples of applications in different microbial communities.
Persistence of a
Salmonella enterica
Serovar Typhimurium DT12 Clone in a Piggery and in Agricultural Soil Amended with
Salmonella
-Contaminated Slurry
Authors: Baloda Suraj B. , Christensen Lise , Trajcevska Silvija
Published: July 2002

Prevalence of
Salmonella enterica
on a Danish pig farm presenting recurrent infections was investigated. A comparison of the pulsed-field gel electrophoresis patterns of fecal isolates from piggeries, waste slurry, and agricultural soil amended with
Salmonella
-contaminated animal waste (slurry) and subclinical isolates from the same farm (collected in 1996 and later) showed identical patterns, indicating long-term persistence of the
Salmonella enterica
serovar Typhimurium DT12 clone in the herd environment. Furthermore, when
Salmonella
-contaminated slurry was disposed of on the agricultural soil (a common waste disposal practice), the pathogen was isolated up to 14 days after the spread, indicating potentially high risks of transmission of the pathogen in the environment, animals, and humans.
Streptomyces
Serine Protease (DHP-A) as a New Biocatalyst Capable of Forming Chiral Intermediates of 1,4-Dihydropyridine Calcium Antagonists
Authors: Arisawa Akira , Matsufuji Motoko , Nakashima Takashi , Dobashi Kazuyuki , Isshiki Kunio , Yoshioka Takeo , Yamada Shigeru , Momose Haruo , Taguchi Seiichi
Published: July 2002

Streptomyces viridosporus
A-914 was screened as a producer of an enzyme to effectively form chiral intermediates of 1,4-dihydropyridine calcium antagonists. The supernatant liquid of the growing culture of this strain exhibited high activity for enantioselective hydrolysis of prochiral 1,4-dihydropyridine diesters to the corresponding (4
R
) half esters. The responsible enzyme (termed DHP-A) was purified to apparent homogeneity and characterized. Cloning and sequence analysis of the gene for DHP-A (
dhpA
) revealed that the enzyme was a serine protease that is highly similar in both structural and enzymatic feature to SAM-P45, which is known as a target enzyme of
Streptomyces
subtilisin inhibitor (SSI), from
Streptomyces albogriseolus
. In a batch reaction test, DHP-A produced a higher yield of a chiral intermediate of 1,4-dihydropyridine than the commercially available protease P6. Homologous or heterologous expression of
dhpA
resulted in overproduction of the enzyme in culture supernatants, with 2.4- to 4.2-fold higher specific activities than in the parent
S. viridosporus
A-914. This indicates that DHP-A is suitable for use in reactions forming chiral intermediates of calcium antagonists and suggests the feasibility of developing DHP-A as a new commercial enzyme for use in the chiral drug industry.
Enhancement by Ampicillin of Antibody Responses Induced by a Protein Antigen and a DNA Vaccine Carried by Live-Attenuated
Salmonella enterica
Serovar Typhi
Authors: Woo Patrick C. Y. , Tsoi Hoi-Wah , Leung Harry C. H. , Wong Lei-Po , Wong Samson S. Y. , Chan Eric , Yuen Kwok-Yung
Published: July 2002

Live-attenuated
Salmonella
species are effective carriers of microbial antigens and DNA vaccines. In a mouse model, the immunoglobulin M (IgM) and total antibody levels directed toward the lipopolysaccharide of
Salmonella enterica
serovar Typhi were significantly enhanced at day 21 after oral immunization with live-attenuated serovar Typhi (strain Ty21a) when ampicillin was concomitantly administered (
P
< 0.05 and
P
< 0.005, respectively). The heat-killed Ty21a-stimulated lymphocyte proliferation indices for the ampicillin group at day 21 were significantly higher than those for the normal saline (NS) group (
P
< 0.005,
P
< 0.001, and
P
< 0.01) for all three doses of antigen (10
4
, 10
5
, and 10
6
heat-killed Ty21a per well, respectively). The 50% lethal doses for mice from the ampicillin and NS groups immunized with Ty21a with pBR322 after wild-type serovar Typhi challenge on day 24 were 3.4 × 10
7
and 5.0 × 10
6
CFU, respectively. The fecal bacterial counts for the ampicillin group at days 1, 3, and 5 were significantly lower than those for the NS group (
P
< 0.01,
P
< 0.01, and
P
< 0.05, respectively), and there was a trend toward recovery of Ty21a in a larger number of mice from the ampicillin group than from the NS group. Furthermore, the IgG2a levels directed toward tetanus toxoid were significantly enhanced at days 7 and 21 after oral immunization with Ty21a that carried the fragment c of tetanus toxoid when ampicillin was concomitantly administered (
P
< 0.05 and
P
< 0.005, respectively), and the IgM and total hepatitis B surface antibody levels were significantly enhanced at days 7 (
P
< 0.005 and
P
< 0.05, respectively) and 21 (
P
< 0.01 and
P
< 0.05, respectively) after oral immunization with Ty21a that carried the DNA vaccine that encodes hepatitis B surface antigen when ampicillin was concomitantly administered. The present observation may improve the efficacy of the protein antigens and DNA vaccines carried in live-attenuated bacteria, and further experiments should be carried out to determine the best antibiotics and dosage regimen to be used, as well as the best carrier system for individual protein antigens and DNA vaccines.
Evaluation of Two Slide Agglutination Tests and a Novel Immunochromatographic Assay for Rapid Diagnosis of Infectious Mononucleosis
Authors: Gómez M. Concepción , Nieto Jose A. , Escribano M. Angeles
Published: July 2002

The results of three tests used for the rapid diagnosis of infectious mononucleosis (IM) were compared with those of Epstein-Barr virus-specific serology. The sensitivities ranged from 15 to 33% in children under 13 years of age and from 59 to 81% in patients over 13 years. The specificities ranged from 86 to 100% in both age groups. These tests have a poor sensitivity for the diagnosis of IM, particularly in children.
Brucella abortus
RB51 and Hot Saline Extract from
Brucella ovis
as Antigens in a Complement Fixation Test Used To Detect Sheep Vaccinated with
Brucella abortus
RB51
Authors: Adone Rosanna , Ciuchini Franco
Published: July 2002

The efficacy of
Brucella abortus
RB51 and hot saline extract (HSE) from
Brucella ovis
as antigens in complement fixation (CF) tests was comparatively evaluated in detecting immune responses of sheep vaccinated with
B. abortus
strain RB51. For this study, four 5-month-old sheep were vaccinated subcutaneously with 5 × 10
9
CFU of RB51, and two sheep received saline. Serum samples collected at different times after vaccination were tested for the presence of antibodies to RB51 by a CF test with RB51 as antigen, previously deprived of anticomplementary activity, and with HSE antigen, which already used as the official antigen to detect
B. ovis
-infected sheep. The results showed that vaccinated sheep developed antibodies which reacted weakly against HSE antigen and these antibodies were detectable for 30 days after vaccination. However, antibodies to RB51 could be detected for a longer period after vaccination by using homologous RB51 antigen in CF tests. In fact, high titers were still present at 110 days postvaccination with RB51 antigen. Sera from sheep naturally infected with
B. ovis
also reacted to RB51 but gave lower titers than those detected by HSE antigen. As expected, all sera from RB51-vaccinated sheep remained negative when tested with standard S-type
Brucella
standard antigens.
Development of a Novel Screen for Protease Inhibitors
Authors: Gillim L. , Gusella G. L. , Vargas J. , Marras D. , Klotman M. E. , Cara A.
Published: July 2002

We have developed a novel plasmid-based, quantitative, in vitro screen to test the protease-inhibiting activities of existing and newly discovered agents.
Comparison of Immulite with Vidas for Detection of Infection in a Low-Prevalence Population of Pregnant Women in The Netherlands
Authors: Vlaspolder F. , Singer P. , Smit A. , Diepersloot R. J. A.
Published: July 2002

A comparative evaluation of the Vidas system (bioMérieux, Marcy l'Etoile, France) and the Immulite System (Diagnostic Products Corporation) was performed using 500 prospectively collected serum samples. As part of a routine antenatal screening program, these samples were tested for hepatitis B surface antigen, and immunoglobulin G (IgG) and IgM antibodies to
Toxoplasma gondii
and rubella virus. The overall agreement between the two assay systems ranged from 98.0 to 99.8%. After discrepancy analysis the outcome in terms of relative sensitivity and specificity varied from 97.5 to 100%.
Intra- and Interlaboratory Variabilities of Results Obtained with the Quantiplex Human Immunodeficiency Virus Type 1 RNA bDNA Assay, Version 3.0
Authors: Kellogg James A. , Atria Peter V. , Sanders Jeffrey C. , Eyster M. Elaine
Published: July 2002

Normal assay variation associated with bDNA tests for human immunodeficiency virus type 1 (HIV-1) RNA performed at two laboratories with different levels of test experience was investigated. Two 5-ml aliquots of blood in EDTA tubes were collected from each patient for whom the HIV-1 bDNA test was ordered. Blood was stored for no more than 4 h at room temperature prior to plasma separation. Plasma was stored at −70°C until transported to the Central Pennsylvania Alliance Laboratory (CPAL; York, Pa.) and to the Hershey Medical Center (Hershey, Pa.) on dry ice. Samples were stored at ≤−70°C at both laboratories prior to testing. Pools of negative (donor), low-HIV-1-RNA-positive, and high-HIV-1-RNA-positive plasma samples were also repeatedly tested at CPAL to determine both intra- and interrun variation. From 11 August 1999 until 14 September 2000, 448 patient specimens were analyzed in parallel at CPAL and Hershey. From 206 samples with results of ≥1,000 copies/ml at CPAL, 148 (72%) of the results varied by ≤0.20 log
10
when tested at Hershey and none varied by >0.50 log
10
. However, of 242 specimens with results of <1,000 copies/ml at CPAL, 11 (5%) of the results varied by >0.50 log
10
when tested at Hershey. Of 38 aliquots of HIV-1 RNA pool negative samples included in 13 CPAL bDNA runs, 37 (97%) gave results of <50 copies/ml and 1 (3%) gave a result of 114 copies/ml. Low-positive HIV-1 RNA pool intrarun variation ranged from 0.06 to 0.26 log
10
while the maximum interrun variation was 0.52 log
10
. High-positive HIV-1 RNA pool intrarun variation ranged from 0.04 to 0.32 log
10
, while the maximum interrun variation was 0.55 log
10
. In our patient population, a change in bDNA HIV-1 RNA results of ≤0.50 log
10
over time most likely represents normal laboratory test variation. However, a change of >0.50 log
10
, especially if the results are >1,000 copies/ml, is likely to be significant.
Characterization of a Monoclonal Antibody That Binds to an Epitope on Soluble Bacterial Peptidoglycan Fragments
Authors: Merkel Glenn J. , Scofield Barbara A.
Published: July 2002

We employed an inhibition-type enzyme-linked immunosorbent assay (ELISA) to characterize a murine immunoglobulin M monoclonal antibody (MAb) that bound soluble macromolecular peptidoglycan (PG). With this ELISA, the MAb was capable of detecting soluble PG concentrations of less than 10 ng/ml. Enzymatic digestion of PG reduced binding by more than 100-fold, implying that the epitope recognized by this antibody depended on repeating subunits within the glycan backbone. Additionally, the MAb bound to epitopes on both O-acetylated and non-O-acetylated PG fragments from gram-negative bacteria, as well as PG fragments from
Staphylococcus aureus
and PG fragments released into the medium by a number of gram-positive and gram-negative bacteria.
Expression of a Novel Protein by Regenerating Hepatocytes and Peripheral Blood Lymphocytes
Authors: Chedid Antonio , Sung Ching C. , Lepe Maria R. , Ahmed Syed A. , Iftikhar Syeda A. , Feller Axel , Beaman Kenneth D.
Published: July 2002

Regeneration and tolerance factor (RTF) is a protein with immunosuppressive activity and is normally present in the thymus and placenta. RTF was measured in the livers of patients with regenerating nodules due to alcoholic cirrhosis and hepatitis C. RTF was expressed in the regenerating nodules of 26 patients with alcoholic cirrhosis. All patients with chronic hepatitis C without cirrhosis failed to express RTF. Flow cytometry revealed upregulation of RTF on the lymphocytes from alcoholic cirrhosis and downregulation in hepatitis C disease.
Immunophenotyping of Peripheral Blood Lymphocytes in Saudi Men
Authors: Al Qouzi Abdulla , Al Salamah Abdulla , Al Rasheed Reem , Al Musalam Abdulla , Al Khairy Khalid , Kheir Osman , Al Ajaji Sulieman , Hajeer Ali H.
Published: July 2002

Flow cytometry is an important tool for the diagnosis and follow-up of immunodeficiency patients, as well as for pateints with leukemia and lymphoma. Lymphocytes and their subsets show variations with race. The aim of this study was to establish reference ranges for lymphocytes and their subsets in an Saudi adult population by using flow cytometry. Blood samples obtained from 209 healthy Saudi men were used for this study. All blood donors were between 18 and 44 years old. Lymphocytes and their subsets were analyzed by flow cytometry, and the absolute and percentage values were calculated. We investigated the expression of T-cell markers (CD3, CD4, and CD8), B cells (CD19), and natural killer cells (CD16 and CD56). The absolute and percent values of each cell subset were compared with published data from different populations by using the Student
t
test. Reference ranges, each expressed as the mean ± the standard deviation, were as follows: leukocytes (6,335 ± 1759), total lymphocytes (2,224 ± 717), CD3 cells (1,618 ± 547), CD4 cells (869 ± 310), CD8 cells (615 ± 278), CD19 cells (230 ± 130), and CD3-CD16
+
/CD56+ cells (262 ± 178). The CD4/CD8 ratio was 1.6 ± 0.7. Our results for B cells, CD4 cells, and CD8 cells and for the CD4/CD8 ratio fell in between the reported results for Ethiopian and Dutch subjects. Our results were also different from previously reported findings in an Saudi adult population that showed no increase in CD8 T cells. We thus establish here the reference ranges for lymphocytes and their subsets in a large cohort of Saudi men. The CD8 cell count was not abnormally high, as previously reported, and fell in between previous results obtained for African and European populations.
Granulocyte Colony-Stimulating Factor Improves Deficient In Vitro Neutrophil Transendothelial Migration in Patients with Advanced Liver Disease
Authors: Fiuza Carmen , Salcedo Magdalena , Clemente Gerardo , Tellado Jose M.
Published: July 2002

Bacterial infections are frequent complications in patients with liver cirrhosis. Cirrhotic patients present abnormalities in both innate and adaptive immune responses, including a deficient neutrophil recruitment to infected sites. The purpose of this study was to assess neutrophil-endothelium interactions in cirrhotic patients and evaluate the effects of G-CSF on this process. We studied neutrophil adhesion and transendothelial migration in 14 cirrhotic patients and 14 healthy controls. We also analyzed neutrophil expression of the adhesion molecules CD62L and CD11b in whole blood by flow cytometry. Cirrhotic patients expressed higher levels of CD11b than healthy controls, whereas CD62L expression was significantly lower, suggesting exposure of neutrophils to activating agents within the bloodstream. Neutrophils from cirrhotic patients showed increased adhesion to both resting and tumor necrosis factor alpha-stimulated microvascular endothelial cells and decreased transendothelial migration. Granulocyte colony-stimulating factor (G-CSF) (100 ng/ml) significantly enhanced neutrophil adhesion to microvascular endothelial cells in healthy controls but not in cirrhotic patients. G-CSF also significantly improved neutrophil transmigration in cirrhotic patients and healthy controls. In conclusion, cirrhotic patients exhibit increased neutrophil adhesion to microvascular endothelium and deficient transendothelial migration. G-CSF enhances neutrophil transendothelial migration in cirrhotic patients despite having no effect on neutrophil adhesion. Therefore, G-CSF may be able to increase neutrophil recruitment into infected sites in these patients.
Reactivity of Rheumatic Fever and Scarlet Fever Patients' Sera with Group A Streptococcal M Protein, Cardiac Myosin, and Cardiac Tropomyosin: a Retrospective Study
Authors: Jones Kevin F. , Whitehead Stephen S. , Cunningham Madeleine W. , Fischetti Vincent A.
Published: July 2002

Archived sera (collected in 1946) from acute rheumatic fever (ARF) and untreated scarlet fever and/or pharyngitis patients were reacted with streptococcal M protein, cardiac myosin, and cardiac tropomyosin. Except for very low levels to tropomyosin, antibodies to other antigens were not elevated in the sera of ARF patients relative to those of non-ARF patients, even though there was roughly equivalent exposure to group A streptococci. This suggests that antibodies to these molecules may not play a central role in the induction of ARF.
Vaccine Efficacy of RecombinantPlasmodium falciparumMerozoite Surface Protein 1 in Malaria-Naive, -Exposed, and/or -RechallengedAotus vociferansMonkeys
Authors: Egan Andrea F. , Blackman Michael J. , Kaslow David C.
Published: July 2002

Protection against a lethal challenge infection ofPlasmodium falciparumwas elicited in malaria-naiveAotus vociferansmonkeys by vaccination with the C terminus 19-kDa protein of the major merozoite surface protein (MSP-119) fused to tetanus toxoid universal T-cell epitopes P30 and P2. Three of four monkeys were protected against a 104-parasite challenge. Four monkeys were challenged with 105parasites; one self-cured the infection, two were protected against high parasitemia (<2%) but were treated for severe anemia (hematocrit of <25%), and the fourth was not protected. In this model system, anemia appears to be a manifestation of incomplete protection (prolonged low-level parasitemia). Enzyme-linked immunosorbent assay (ELISA) antibody titers correlated with protection. Antibodies from some protected monkeys inhibited secondary processing of MSP-142to MSP-133and MSP-119. To mimic the repeated reinfections seen in regions where malaria is endemic, a second malaria parasite challenge was administered 4 months later. All P30P2MSP-119-vaccinated monkeys were protected; thus, a single challenge infection may underestimate vaccine efficacy. ELISA antibody titers correlated with protection against a second infection but had decreased compared to the first challenge. As most target populations for asexual blood-stage malaria vaccines will have been exposed to malaria parasites, a malaria parasite-exposed monkey was vaccinated with P30P2MSP-119. This monkey was completely protected, while a malaria parasite-naive P30P2MSP-119-vaccinated monkey self-cured a low-grade parasitemia. Prior malaria parasite infection primed the production of anti-native MSP-119antibodies, which were boosted by vaccination with recombinant P30P2MSP-119. Preliminary data suggest that immunogenicity studies of vaccines designed for malaria parasite-exposed populations should also be conducted in malaria parasite-exposed subjects.
RpmA Is Required for Nonopsonic Phagocytosis of
Pseudomonas aeruginosa
Authors: Simpson David A. , Speert David P.
Published: July 2002

Pseudomonas aeruginosa
causes severe respiratory tract infections in patients with cystic fibrosis (CF). We have been examining nonopsonic phagocytosis of
P. aeruginosa
by macrophages. To study the
P. aeruginosa
-macrophage interaction at the molecular level, we have constructed a transposon Tn
5
G bank in a clinical isolate of
P. aeruginosa
(strain 4020) and identified mutants resistant to nonopsonic phagocytosis. Phagocytosis-resistant mutants were enriched by passaging the transposon bank over 18 macrophage monolayers. Of 900 individual mutants isolated from this enriched pool in a nonopsonic phagocytosis assay, we identified 85 putative mutants that were resistant to phagocytosis. In this study, we have characterized one of these transposon mutants,
P. aeruginosa
4020 H27A, which was poorly ingested. H27A possessed a Tn
5
G insertion in a gene encoding a protein with homology to the MotA proteins of several species of bacteria. We have called this gene
rpmA
for required for phagocytosis by macrophages. RpmA is one of two MotA paralogs in
P. aeruginosa
. This
rpmA
::Tn
5
G mutant was motile both on agar plates and in visual examination of wet mounts. The phagocytosis defect was partially complemented by providing the
rpmA
gene in
trans
and fully complemented when both
rpmA
and
rpmB
were provided. A
rpmA
null mutant was ingested by macrophages similar to the H27A transposon mutant. These data suggest that the
rpmA
and
rpmB
gene products are required for the efficient ingestion of
P. aeruginosa
by macrophages.
Coimmunization with Complementary Glucosyltransferase Peptides Results in Enhanced Immunogenicity and Protection against Dental Caries
Authors: Taubman Martin A. , Smith Daniel J. , Holmberg Cynthia J. , Eastcott Jean W.
Published: July 2002

Peptide constructs from the catalytic (CAT) and glucan-binding (GLU) regions of the mutans streptococcal glucosyltransferase enzymes (GTF) can provide immunity to dental caries infection. A strategy of coimmunization was tested to determine whether protection could be enhanced. Rats were immunized with one of the previously described peptide constructs from the CAT or GLU region of the GTF of mutans streptococci or coimmunized with a combination of these constructs (CAT-GLU). Coimmunized animals demonstrated significantly higher serum immunoglobulin G (IgG) and salivary IgA antibody levels to CAT or GTF than rats immunized with either construct alone. To assess the functional significance of coimmunization with these constructs, animals were immunized as above or with
Streptococcus sobrinus
GTF and then infected with
S. sobrinus
to explore the effects of immunization on immunological, microbiological, and disease (dental caries) parameters. Serum antibody from the communized group inhibited
S. sobrinus
GTF-mediated insoluble glucan synthesis in vitro above that of the individual-construct-immunized groups. Immunization with CAT or GLU constructs resulted in significantly reduced dental caries after infection with
S. sobrinus
compared with sham-immunized animals. Coimmunization produced greater reductions in caries than after immunization with either CAT or GLU. Also, significant elevations in lymphocyte proliferative responses to CAT, GLU, and GTF were observed after coimmunization with CAT-GLU compared with the responses after immunization with the individual constructs. The results suggested that increased numbers of memory T cells, which could proliferate to CAT, were generated by coimmunization. The experiments support the functional significance of these GTF domains in dental caries pathogenesis and present coimmunization as a simple alternative to intact GTF to enhance protective immunity against cariogenic microorganisms.
Functional Analysis of Genes in the
rfb
Locus of
Leptospira borgpetersenii
Serovar Hardjo Subtype Hardjobovis
Authors: Bulach Dieter M. , Kalambaheti Thareerat , de la Peña-Moctezuma Alejandro , Adler Ben
Published: July 2002

Lipopolysaccharide (LPS) is a key antigen in immunity to leptospirosis. Its biosynthesis requires enzymes for the biosynthesis and polymerization of nucleotide sugars and the transport through and attachment to the bacterial membrane. The genes encoding these functions are commonly clustered into loci; for
Leptospira borgpetersenii
serovar Hardjo subtype Hardjobovis, this locus, named
rfb
, spans 36.7 kb and contains 31 open reading frames, of which 28 have been assigned putative functions on the basis of sequence similarity. Characterization of the function of these genes is hindered by the fact that it is not possible to construct isogenic mutant strains in
Leptospira
. We used two approaches to circumvent this problem. The first was to clone the entire locus into a heterologous host system and determine if a “recombinant” LPS or polysaccharide was synthesized in the new host. The second approach used putative functions to identify mutants in other bacterial species whose mutations might be complemented by genes on the leptospiral
rfb
locus. This approach was used to investigate the function of three genes in the leptospiral
rfb
locus and demonstrated function for
orfH10
, which complemented a
wbpM
strain of
Pseudomonas aeruginosa
, and
orfH13
, which complemented an
rfbW
strain of
Vibrio cholerae
. However, despite the similarity of OrfH11 to WecC, a
wecC
strain of
E. coli
was not complemented by
orfH11
. The predicted protein encoded by
orfH8
is similar to GalE from a number of organisms. A
Salmonella enterica
serovar Typhimurium strain producing no GalE was used as a background in which
orfH8
produced detectable GalE enzyme activity.
Pseudomonas aeruginosaExoenzyme S Induces Transcriptional Expression of Proinflammatory Cytokines and Chemokines
Authors: Epelman Slava , Bruno Tony F. , Neely Graham G. , Woods Donald E. , Mody Christopher H.
Published: July 2002

Pseudomonas aeruginosainfection of cystic fibrosis patients causes lung damage that is substantially orchestrated by cytokines. In this study, multi-gene probe analysis was used to characterize the ability of theP. aeruginosamitogen, exoenzyme S, to induce proinflammatory and immunoregulatory cytokines and chemokines. Exoenzyme S strongly induced transcription of proinflammatory cytokines and chemokines (tumor necrosis factor alpha, interleukin-1α [IL-1α], IL-1β, IL-6, IL-8, MIP-1α, MIP-1β, MCP-1, RANTES, and I-309), modest transcription of immunoregulatory cytokines (IL-10 and IL-12p40), and weak transcription of Th1 cytokines (IL-2 and gamma interferon). The response occurred early and subsided without evolving over time. These data suggest that cells responding to exoenzyme S would rapidly express proinflammatory cytokines and chemokines that may contribute to pulmonary inflammation in cystic fibrosis.
Neisseria gonorrhoeaeElicits Membrane Ruffling and Cytoskeletal Rearrangements upon Infection of Primary Human Endocervical and Ectocervical Cells
Authors: Edwards Jennifer L. , Shao Jian Q. , Ault Kevin A. , Apicella Michael A.
Published: July 2002

Neisseria gonorrhoeaeis a strict human pathogen that is, primarily, transmitted by close sexual contact with an infected individual. Gonococcal infection of the male urogenital tract has been well studied in experimental human models and in urethral cell culture systems. Recent studies, using tissue culture cell systems, have suggested a role for the cervical epithelium in gonococcal infection of females; however, the nature of gonococcal infection of the normal uterine cervix remains controversial. To address this enigma, we have developed two primary human cervical epithelial cell systems from surgical biopsies. Gonococcal infection studies and electron microscopy show thatN. gonorrhoeaeis capable of infecting and invading both the endo- and the ectocervix. Invasion was found to occur primarily in an actin-dependent manner, but it does not appear to require de novo protein synthesis by either the bacterium or the host cervical cell. Membrane ruffles appear to be induced in response to gonococci. Consistent with membrane ruffling, gonococci were found residing within macropinosomes, and a concentrated accumulation of actin-associated proteins was observed to occur in response to gonococcal infection. Electron microscopy of clinically derived cervical biopsies show that lamellipodia formation and cytoskeletal changes, suggestive of membrane ruffles, also occur in the cervical epithelium of women with naturally acquired gonococcal cervicitis. These studies demonstrate the ability ofN. gonorrhoeaeto infect and invade both the endo- and the ectocervix of the normal uterine cervix. Gonococcal induced ruffling is a novel finding and may be unique to the cervical epithelium.
Mouse Toxicity and Cytokine Release by Verotoxin 1 B Subunit Mutants
Authors: Wolski Vince M. , Soltyk Anna M. , Brunton James L.
Published: July 2002

The crystal structure of the verotoxin 1 (VT1) B subunit complexed with a globotriaosylceramide (Gb
3
) analogue showed the presence of three receptor binding sites per monomer. We wished to study the effects of altering the three sites, singly or in combination, on animal toxicity and cytokine induction in vitro. We found that while the site 1 and 2 mutants were modestly (two- to sevenfold) reduced in their ability to cause disease in BALB/c mice, the site 3 mutant, W34A, was as toxic as VT1. However, all the double-mutant proteins, irrespective of which two sites were mutated, exhibited approximately a 100-fold reduction in their 50% lethal doses for mice. These results suggest that multivalent receptor binding is important in vivo and that all three binding sites make a similar contribution to the latter process. The triple-mutant holotoxin, F30A G62T W34A, administered intraperitoneally without adjuvant, stimulated a strong antibody response in BALB/c mice, and the immune sera neutralized the activity of VT1 in vitro. Induction of tumor neurosis factor alpha release from differentiated human monocytes (THP-1 cells) was relatively impaired for site 1 and site 2 but not site 3 mutants, suggesting an auxiliary role for the latter site in mediation of cytokine release in vitro. Cytotoxicity assays on undifferentiated THP-1 cells have also demonstrated the importance of sites 1 and 2 and the relatively small role played by site 3 in causing cell death. These data suggest an association between the cytotoxicity of the protein and its ability to induce cytokine release.
Induction of Protective Immunity againstStreptococcus mutansColonization after Mucosal Immunization with AttenuatedSalmonella entericaSerovar Typhimurium Expressing anS. mutansAdhesin under the Control of In Vivo-InduciblenirBPromoter
Authors: Huang Yan , Hajishengallis George , Michalek Suzanne M.
Published: July 2002

The purpose of the present study was to evaluate the effectiveness of an attenuatedSalmonella entericaserovar Typhimurium vaccine strain expressing the saliva-binding region (SBR) of theStreptococcus mutansantigen I/II adhesin, either alone or linked with the mucosal adjuvant cholera toxin A2 and B subunits (CTA2/B) and under the control of the anaerobically induciblenirBpromoter, in inducing a protective immune response againstS. mutansinfection. BALB/c mice were immunized by either the intranasal or the intragastric route with a single dose of 109or 1010SalmonellaCFU, respectively. TheSalmonellavaccine strain expressing an unrelated antigen (fragment C of tetanus toxin [TetC]) was also used for immunization as a control. Samples of serum and secretion (saliva and vaginal washes) were collected prior to and following immunization and assessed for antibody activity by enzyme-linked immunosorbent assay. Anti-SBR antibodies were detected in the serum and saliva of experimental animals by week 3 after immunization. A booster immunization at week 17 after the initial immunization resulted in enhanced immune responses to the SBR. The serum immunoglobulin G subclass profiles were indicative of T helper type 1 responses against both the vector and the SBR antigen. To determine the effectiveness of these responses on the protection againstS. mutansinfection, mice were challenged after the second immunization with a virulent strain ofS. mutanswhich was resistant to tetracycline and erythromycin. Prior to the challenge, mice were treated for 5 days with tetracycline, erythromycin, and penicillin.S. mutanswas initially recovered from all of the challenged mice. This bacterium persisted at high levels for at least 5 weeks in control TetC-immunized or nonimmunized mice despite the reappearance of indigenous oral organisms. However, mice immunized withSalmonellaclones expressing SBR or SBR-CTA2/B demonstrated a significant reduction in the number ofS. mutanspresent in plaque compared to the control groups. These results provide evidence for the effectiveness of theSalmonellavector in delivering the SBR antigen for the induction of mucosal and systemic immune responses to SBR. Furthermore, the induction of a salivary anti-SBR response corresponded with protection againstS. mutanscolonization of tooth surfaces.
Ureaplasma urealyticumModulates Endotoxin-Induced Cytokine Release by Human Monocytes Derived from Preterm and Term Newborns and Adults
Authors: Manimtim Winston M. , Hasday Jeffrey D. , Hester Lisa , Fairchild Karen D. , Lovchik Judith C. , Viscardi Rose M.
Published: July 2002

We previously observed thatUreaplasma urealyticumrespiratory tract colonization in infants with a birth weight of ≤1,250 g was associated with increases in the tracheal aspirate proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-8 (IL-8) relative to the counterregulatory cytokine IL-6 during the first week of life (A. M. Patterson, V. Taciak, J. Lovchik, R. E. Fox, A. B. Campbell, and R. M. Viscardi, Pediatr. Infect. Dis. J. 17:321–328, 1998). We hypothesized thatU. urealyticumalters the host immune response in the presence of a coinflammatory stimulus (e.g., bacterial infection or hyperoxia) by shifting the balance of cytokine expression towards the proinflammatory cytokines. To test this hypothesis, we compared the release of TNF-α, IL-8, IL-6, and IL-10 in vitro by unstimulated andU. urealyticum(with or without lipopolysaccharide [LPS])-stimulated human monocytes from adult peripheral blood and from term and preterm cord blood.U. urealyticumalone and in combination with LPS induced concentration- and development-dependent changes in cytokine release. In vitro inoculation with low-inoculumU. urealyticum(103color-changing units [CCU]) (i) partially blocked the LPS-stimulated IL-6 release by all cells and reduced LPS-stimulated IL-10 release by preterm cells, (ii) stimulated TNF-α and IL-8 release by preterm cells, and (iii) augmented LPS-stimulated TNF-α release in all cells. In preterm cells, high-inoculumU. urealyticum(106CCU) (i) stimulated TNF-α and IL-8, but not IL-6 or IL-10, release and (ii) augmented LPS-stimulated TNF-α and IL-8 release. High-inoculumU. urealyticum(i) stimulated release of all four cytokines in term cells and IL-8 release in adult cells and (ii) augmented LPS-induced TNF-α, IL-10, and IL-8 release in term cells but did not significantly affect LPS-induced cytokine release in adult cells. We speculate thatU. urealyticumenhances the proinflammatory response to a second infection by blocking expression of counterregulatory cytokines (IL-6 and IL-10), predisposing the preterm infant to prolonged and dysregulated inflammation, lung injury, and impaired clearance of secondary infections.
Neisseria meningitidis
RTX Protein FrpC Induces High Levels of Serum Antibodies during Invasive Disease: Polymorphism of
frpC
Alleles and Purification of Recombinant FrpC
Authors: Osička Radim , Kalmusová Jitka , Křı́žová Pavla , Šebo Peter
Published: July 2002

The
Neisseria meningitidis
FAM20 strain secretes two proteins of unknown function, FrpA and FrpC, which contain typical RTX domains found in cytotoxins from other gram-negative pathogens. To evaluate whether the Frp proteins could be involved in meningococcal virulence, 65 isolates of all serogroups were screened by PCR for the presence of both
frp
genes. The
frpA
allele was, however, poorly conserved. A single strain harbored an
frpA
allele of the previously described size, while large insertions were detected in the
frpA
loci of 22 isolates (34%), and the 42 remaining isolates (65%) did not contain
frpA
at all. In contrast,
frpC
alleles, albeit of variable length, were detected in all invasive and most carrier strains. This suggests that meningococci may produce a family of FrpC proteins of various molecular masses. High levels of both immunoglobulin G (IgG) and IgA class antibodies recognizing recombinant FrpC were indeed detected in convalescent-phase sera of most patients at 2 and 4 to 5 weeks after the first symptoms of meningococcal disease. These results show that FrpC-like proteins are produced and may play a role in invasive meningococcal infections.
Central Role for Interleukin-4 in Regulating Nitric Oxide-Mediated Inhibition of T-Cell Proliferation and Gamma Interferon Production in Schistosomiasis
Authors: Patton Elisabeth A. , La Flamme Anne C. , Pedras-Vasoncelos Joao A. , Pearce Edward J.
Published: July 2002

Schistosoma mansoni-infected wild-type (WT) mice develop a Th2 response and chronic disease. In contrast, infected interleukin-4 double-deficient (IL-4−/−) mice develop a Th1-like response and an acute, lethal syndrome. Disease severity in these animals correlates with excessive and prolonged production of nitric oxide (NO) associated with enhanced antigen-driven gamma interferon (IFN-γ) production in the absence of IL-4. Strikingly, splenic lymphocytes from infected IL-4−/−mice failed to proliferate as well as those from infected WT mice following stimulation in vitro with antigen or anti-CD3 antibody. Contrary to antigen-driven IFN-γ responses, anti-CD3 antibody stimulation of splenocytes resulted in significantly less IFN-γ being produced by CD8 cells from infected IL-4−/−mice than by those from infected WT mice or normal mice. NO is largely responsible for the impaired T-cell functions in infected IL-4−/−mice, as inhibition of iNOS significantly enhanced proliferation and IFN-γ production.
Recombinant Tumor Necrosis Factor Alpha Does Not Inhibit the Growth of African Trypanosomes in Axenic Cultures
Authors: Kitani Hiroshi , Black Samuel J. , Nakamura Yoshio , Naessens Jan , Murphy Noel B. , Yokomizo Yuichi , Gibson John , Iraqi Fuad
Published: July 2002

Mice whose tumor necrosis factor alpha (TNF-α) genes were disrupted developed higher levels of parasitemia than wild-type mice following infection with
Trypanosoma congolense
IL1180 or
T. brucei brucei
GUTat3.1, confirming the results of earlier studies. To determine whether TNF-α directly affects the growth of these and other bloodstream forms of African trypanosomes, we studied the effects of recombinant mouse, human, and bovine TNF-α on the growth of two isolates of
T. congolense
, IL1180 and IL3338, and two isolates of
T. brucei brucei
, GUTat3.1 and ILTat1.1, under axenic culture conditions. The preparations of recombinant TNF-α used were biologically active as determined by their capacity to kill L929 cells. Of five recombinant TNF-α lots tested, one lot of mouse TNF-α inhibited the growth of both isolates of
T. brucei brucei
and one lot of bovine TNF-α inhibited the growth of
T. brucei brucei
ILTat1.1 but only at very high concentrations and without causing detectable killing of the parasites. The other lots of mouse recombinant TNF-α, as well as human TNF-α, did not affect the growth of any of the test trypanosomes even at maximal concentrations that could be attained in the culture systems (3,000 to 15,000 U of TNF-α/ml of medium). These results suggest that exogenously added recombinant TNF-α generally does not inhibit the growth of African trypanosomes under the culture conditions we used. The impact of TNF-α on trypanosome parasitemia may be indirect, at least with respect to the four strains of trypanosomes reported here.
A Lytic Transglycosylase of
Neisseria gonorrhoeae
Is Involved in Peptidoglycan-Derived Cytotoxin Production
Authors: Cloud Karen A. , Dillard Joseph P.
Published: July 2002

Neisseria gonorrhoeae
releases soluble fragments of peptidoglycan during growth. These molecules are implicated in the pathogenesis of various forms of gonococcal infection. A major peptidoglycan fragment released by gonococci is identical to the tracheal cytotoxin of
Bordetella pertussis
and has been shown to kill ciliated fallopian tube cells in organ culture. Previous studies indicated that a unique lytic peptidoglycan transglycosylase (AtlA) was responsible for some, but not all, of the peptidoglycan-derived cytotoxin (PGCT) production in certain gonococcal strains. To examine the role of other putative lytic transglycosylases in PGCT production, we made a deletion mutation in a gonococcal gene exhibiting similarity with genes encoding lytic transglycosylases from other bacterial species. The gonococcal mutant was viable and grew normally, but it was less autolytic than the wild-type strain in stationary-phase culture and under nongrowth conditions. The gonococcal mutant was reduced in peptidoglycan turnover, and the profile of the released products showed a reduction in monomeric peptidoglycan. Proportionally more multimeric fragments were released. These results suggest that this gonococcal gene (
ltgA
) encodes a lytic peptidoglycan transglycosylase and that it is responsible for a significant proportion of the PGCT released by
N. gonorrhoeae
.