bluuebunny/crossref_metadata_2025 huggingface dataset:
Homology of
Escherichia coli
R773
arsA
,
arsB
, and
arsC
Genes in Arsenic-Resistant Bacteria Isolated from Raw Sewage and Arsenic-Enriched Creek Waters
Authors: Saltikov Chad W. , Olson Betty H.
Published: July 2002

The occurrence and diversity of the
Escherichia coli
R773
ars
operon were investigated among arsenic-resistant enteric and nonenteric bacteria isolated from raw sewage and arsenic-enriched creek waters. Selected isolates from each creek location were screened for
ars
genes by colony hybridization and PCR. The occurrence of
arsA
,
arsB
, and
arsC
determined by low-stringency colony hybridization (31 to 53% estimated mismatch) was 81, 87, and 86%, respectively, for 84 bacteria isolated on arsenate- and arsenite-amended media from three locations. At moderate stringency (21 to 36% estimated mismatch), the occurrence decreased to 42, 56, and 63% for
arsA
,
arsB
, and
arsC
, respectively. PCR results showed that the
ars
operon is conserved in some enteric bacteria isolated from creek waters and raw sewage. The occurrence of the
arsBC
genotype was about 50% in raw sewage enteric bacteria, while
arsA
was detected in only 9.4% of the isolates (
n
= 32). The
arsABC
and
arsBC
genotypes occurred more frequently in enteric bacteria isolated from creek samples: 71.4 and 85.7% (
n
= 7), respectively. Average sequence divergence within
arsB
for six creek enteric bacteria was 20% compared to that of the
E. coli
R773
ars
operon. Only 1 of 11 pseudomonads screened by PCR was positive for
arsB
. The results from this study suggest that significant divergence has occurred in the
ars
operon among As-resistant
E. coli
strains and in
Pseudomonas
spp.
Partitioning of Bacterial Communities between Seawater and Healthy, Black Band Diseased, and Dead Coral Surfaces
Authors: Frias-Lopez Jorge , Zerkle Aubrey L. , Bonheyo George T. , Fouke Bruce W.
Published: July 2002

Distinct partitioning has been observed in the composition and diversity of bacterial communities inhabiting the surface and overlying seawater of three coral species infected with black band disease (BBD) on the southern Caribbean island of Curaçao, Netherlands Antilles. PCR amplification and sequencing of bacterial 16S rRNA genes (rDNA) with universally conserved primers have identified over 524 unique bacterial sequences affiliated with 12 bacterial divisions. The molecular sequences exhibited less than 5% similarity in bacterial community composition between seawater and the healthy, black band diseased, and dead coral surfaces. The BBD bacterial mat rapidly migrates across and kills the coral tissue. Clone libraries constructed from the BBD mat were comprised of eight bacterial divisions and 13% unknowns. Several sequences representing bacteria previously found in other marine and terrestrial organisms (including humans) were isolated from the infected coral surfaces, including
Clostridium
spp.,
Arcobacter
spp.,
Campylobacter
spp.,
Cytophaga fermentans
,
Cytophaga columnaris
, and
Trichodesmium tenue
.
Rapid and Quantitative Detection of the Microbial Spoilage of Meat by Fourier Transform Infrared Spectroscopy and Machine Learning
Authors: Ellis David I. , Broadhurst David , Kell Douglas B. , Rowland Jem J. , Goodacre Royston
Published: July 2002

Fourier transform infrared (FT-IR) spectroscopy is a rapid, noninvasive technique with considerable potential for application in the food and related industries. We show here that this technique can be used directly on the surface of food to produce biochemically interpretable “fingerprints.” Spoilage in meat is the result of decomposition and the formation of metabolites caused by the growth and enzymatic activity of microorganisms. FT-IR was exploited to measure biochemical changes within the meat substrate, enhancing and accelerating the detection of microbial spoilage. Chicken breasts were purchased from a national retailer, comminuted for 10 s, and left to spoil at room temperature for 24 h. Every hour, FT-IR measurements were taken directly from the meat surface using attenuated total reflectance, and the total viable counts were obtained by classical plating methods. Quantitative interpretation of FT-IR spectra was possible using partial least-squares regression and allowed accurate estimates of bacterial loads to be calculated directly from the meat surface in 60 s. Genetic programming was used to derive rules showing that at levels of 10
7
bacteria·g
−1
the main biochemical indicator of spoilage was the onset of proteolysis. Thus, using FT-IR we were able to acquire a metabolic snapshot and quantify, noninvasively, the microbial loads of food samples accurately and rapidly in 60 s, directly from the sample surface. We believe this approach will aid in the Hazard Analysis Critical Control Point process for the assessment of the microbiological safety of food at the production, processing, manufacturing, packaging, and storage levels.
Fluorescence In Situ Hybridization and Catalyzed Reporter Deposition for the Identification of Marine Bacteria
Authors: Pernthaler Annelie , Pernthaler Jakob , Amann Rudolf
Published: July 2002

Fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification, also known as catalyzed reporter deposition (CARD), is currently not generally applicable to heterotrophic bacteria in marine samples. Penetration of the HRP molecule into bacterial cells requires permeabilization procedures that cause high and most probably species-selective cell loss. Here we present an improved protocol for CARD-FISH of marine planktonic and benthic microbial assemblages. After concentration of samples onto membrane filters and subsequent embedding of filters in low-gelling-point agarose, no decrease in bacterial cell numbers was observed during 90 min of lysozyme incubation (10 mg ml
−1
at 37°C). The detection rates of coastal North Sea bacterioplankton by CARD-FISH with a general bacterial probe (EUB338-HRP) were significantly higher (mean, 94% of total cell counts; range, 85 to 100%) than that with a monolabeled probe (EUB338-mono; mean, 48%; range, 19 to 66%). Virtually no unspecific staining was observed after CARD-FISH with an antisense EUB338-HRP. Members of the marine SAR86 clade were undetectable by FISH with a monolabeled probe; however, a substantial population was visualized by CARD-FISH (mean, 7%; range, 3 to 13%). Detection rates of EUB338-HRP in Wadden Sea sediments (mean, 81%; range, 53 to 100%) were almost twice as high as the detection rates of EUB338-mono (mean, 44%; range, 25 to 71%). The enhanced fluorescence intensities and signal-to-background ratios make CARD-FISH superior to FISH with directly labeled oligonucleotides for the staining of bacteria with low rRNA content in the marine environment.
Conjugation of Hydroxyethyl Starch to Desferrioxamine (DFO) Modulates the Dual Role of DFO in
Yersinia enterocolitica
Infection
Authors: Schubert Sören , Autenrieth Ingo B.
Published: July 2002

The iron chelator desferrioxamine (DFO) B is widely used in the therapy of patients with iron overload. As a side effect, DFO may favor the occurrence of fulminant
Yersinia
infections. Previous work from our laboratory showed that this might be due to a dual role of DFO: growth promotion of the pathogen and immunosuppression of the host. In this study, we sought to determine whether conjugation of DFO to hydroxyethyl starch (HES-DFO) may prevent exacerbation of
Yersinia
infection in mice. We found HES-DFO to promote neither growth of
Yersinia enterocolitica
nor mitogen-induced T-cell proliferation and gamma interferon production by T cells in vitro. Nevertheless, in vivo HES-DFO promoted growth of
Y. enterocolitica
possibly due to cleavage of HES and release of DFO. The pretreatment of mice with DFO resulted in death of all mice 2 to 5 days after application of a normally sublethal inoculum of
Y. enterocolitica
, while none of the mice pretreated with HES-DFO died within the first 7 days postinfection. However, some of the HES-DFO-treated mice died 8 to 14 days postinfection. Thus, due to the delayed in vivo effect HES-DFO failed to trigger
Yersinia
-induced septic shock, which accounts for early mortality in DFO-associated septicemia. Moreover, our data suggest that DFO needs to be taken up by host cells in order to exert its immunosuppressive action. These results strongly suggest that HES-DFO might be a favorable drug with fewer side effects than DFO in terms of DFO-promoted fulminant infections.
Comparison of Two Commercial Microimmunofluorescence Kits and an Enzyme Immunoassay Kit for Detection of Serum Immunoglobulin G Antibodies to
Chlamydia pneumoniae
Authors: Messmer Trudy O. , Martinez Joseph , Hassouna Fadwa , Zell Elizabeth R. , Harris Wayne , Dowell Scott , Carlone George M.
Published: July 2002

We compared the MRL and the Labsystems
Chlamydia pneumoniae
microimmunofluorescence (MIF) immunoglobulin G (IgG) kits and the Labsystems enzyme immunoassay (EIA) kit in a blinded study of 83 serum samples in which we evaluated titers, cross-reactivity to other species, and reproducibility. There was no statistically significant difference between the MRL and the Labsystems MIF kits in the endpoint titers of IgG antibody to
C. pneumoniae
. The correlation between the results obtained with these two MIF kits was excellent (
r
= 0.95;
P
= 0.001). The cross-reactivity of the
C. pneumoniae
-positive sera with
C. trachomatis
- and
C. psittaci
-positive sera was assessed for each MIF kit. For
C. pneumoniae
-positive sera with titers of ≥32, the Labsystems MIF kit exhibited more cross-reactivity to
C. psittaci
than the MRL kit did. The values obtained with the Labsystems EIA kit represented single dilutions of serum specimens expressed as enzymeimmuno units on a continuous scale. The results obtained with the Labsystems EIA kit correlated moderately well with those obtained with each MIF kit when they were compared for their abilities to detect IgG antibodies to
C. pneumoniae
(for the MRL MIF kit,
r
= 0.79 [
P
= 0.001]; for the Labsystems MIF kit,
r
= 0.78 [
P
= 0.001]). The results obtained with the commercial MRL and Labsystems MIF kits and the Labsystems EIA kit tested were reproducible; and the kits were standardized, had quality control reagents, and are suitable for detection of
C. pneumoniae
antibodies in serum and for use in interlaboratory studies. Validation of the use of these kits for clinical diagnosis still needs further evaluation.
Gamma Interferon Expression in CD8+T Cells Is a Marker for Circulating Cytotoxic T Lymphocytes That Recognize an HLA A2-Restricted Epitope of Human Cytomegalovirus Phosphoprotein pp65
Authors: Ghanekar Smita A. , Nomura Laurel E. , Suni Maria A. , Picker Louis J. , Maecker Holden T. , Maino Vernon C.
Published: July 2002

Antigen-specific CD8+T cells with cytotoxic activity are often critical in immune responses to infectious pathogens. To determine whether gamma interferon (IFN-γ) expression is a surrogate marker for cytotoxic T lymphocytes (CTL), human cytomegalovirus-specific CTL responses were correlated with CD8+T-cell IFN-γ expression determined by cytokine flow cytometry. A strong positive correlation was observed between specific lysis of peptide-pulsed targets in a51Cr release assay and frequencies of peptide-activated CD8+T cells expressing IFN-γ at 6 h (r2= 0.72) or 7 days (r2= 0.91). Enumeration of responding cells expressing perforin, another marker associated with CTL, did not improve this correlation. These results demonstrate that IFN-γ expression can be a functional surrogate for identification of CTL precursor cells.
Comparison of Hepatitis C Viral Loads in Patients with or without Human Immunodeficiency Virus
Authors: Matthews-Greer Janice M. , Caldito Gloria C. , Adley Sharon D. , Willis Regina , Mire Angela C. , Jamison Richard M. , McRae Kenny L. , King John W. , Chang Wun-Ling
Published: July 2002

A better understanding of how human immunodeficiency virus (HIV) coinfection affects the course of hepatitis C virus (HCV) infection is required to select patients with HIV who would benefit from current HCV therapy. Between June 1996 and March 2000, HCV RNA levels were quantified for 1,279 patients at the Louisiana State University Health Sciences Center; 28 of these patients were coinfected with HIV. HCV loads were quantified by the Bayer branched-DNA assay with a lower limit of detection of 0.2 Meq/ml. We compared the median HCV RNA levels of for patients coinfected with HIV and HCV and patients infected only with HCV who were in the same age range (23 to 55 years). The median HCV load for the 28 patients coinfected with HCV and HIV (17.8 Meq/ml) was significantly greater (
P
< 0.05) than that for similarly aged patients infected only with HCV (6.1 Meq/ml). The HCV load did not correlate with age or sex for either group of patients. A significant (
R
= −0.4;
P
< 0.05) negative correlation was observed between HCV load and CD4 count in the coinfected group, for whom the CD4 counts at the time of HCV load analysis ranged from 6 to 1,773/mm
3
. The increased HCV load in patients coinfected with HCV and HIV compared to that in patients infected only with HCV and the inverse relationship of the HCV load to the CD4 count indicate that immunosuppression results in decreased control of HCV replication. In addition, we report significantly higher HCV loads among coinfected African Americans than Caucasians.
Excretory-Secretory Antigens of
Trypanosoma cruzi
Are Potentially Useful for Serodiagnosis of Chronic Chagas' Disease
Authors: Nakazawa Mineo , Rosa Daniela S. , Pereira Valéria R. A. , Moura Milena O. , Furtado Veridiana C. , Souza Wayner V. , Barros Maria Das Neves D. S. , Abath Frederico G. C. , Gomes Yara M.
Published: July 2002

The reactivities of sera from chronic chagasic patients against the trypomastigote excreted-secreted antigens (TESA) of
Trypanosoma cruzi
strains with different biodemes were analyzed by TESA-blot and TESA–enzyme-linked immunosorbent assay (ELISA). Although both tests presented high sensitivity and specificity, TESA-ELISA is more appropriate for screening a larger number of samples.
Prevalence of Hepatitis E Virus Antibodies in Canadian Swine Herds and Identification of a Novel Variant of Swine Hepatitis E Virus
Authors: Yoo Dongwan , Willson Philip , Pei Yanlong , Hayes M. Anthony , Deckert Anne , Dewey Catherine E. , Friendship Robert M. , Yoon Yungho , Gottschalk Marcelo , Yason Carmencita , Giulivi Antonio
Published: July 2002

Swine hepatitis E virus is a newly identified potentially zoonotic virus from pigs of particular concern for possible direct transmission to a human xenotransplant recipient by organ transplantation. In the present study, prevalence of serum antibodies to hepatitis E virus was examined in Canadian swine herds. A total of 998 serum samples collected from 6-month-old healthy slaughter hogs were examined by enzyme immunoassay and Western blot analysis for antibodies to the recombinant open reading frame 3 (ORF3) protein of hepatitis E virus expressed in
Escherichia coli
. These samples represented more than 80 different swine production units from five major swine-producing provinces across Canada. From this study, 594 samples (59.4%) were found to be positive for hepatitis E virus antibody. The seroprevalence was higher in Quebec (88.8%) and Ontario (80.1%) than in Alberta and Saskatchewan (38.3%). By PCR using a pair of oligonucleotide primers deduced from the ORF2 sequence of human hepatitis E virus, a specific hepatitis E virus sequence was recovered from feces of pigs. The nucleotide sequence identity between the U.S. swine hepatitis E virus and the Canadian isolate (SK3) was only 85.8%, suggesting that genotypic variations may exist in swine hepatitis E virus in North America. Among 165 serum samples collected from humans in Saskatchewan, 2.4% were found to be positive for antibodies to the hepatitis E virus ORF3 protein. Our data indicate that hepatitis E virus is highly prevalent in commercial swine populations in Canada and support the suggestion that the swine hepatitis E virus may be an important zoonotic agent for humans.
Wound Microbiology and Associated Approaches to Wound Management
Authors: Bowler P. G. , Duerden B. I. , Armstrong D. G.
Published: July 2002

The majority of dermal wounds are colonized with aerobic and anaerobic microorganisms that originate predominantly from mucosal surfaces such as those of the oral cavity and gut. The role and significance of microorganisms in wound healing has been debated for many years. While some experts consider the microbial density to be critical in predicting wound healing and infection, others consider the types of microorganisms to be of greater importance. However, these and other factors such as microbial synergy, the host immune response, and the quality of tissue must be considered collectively in assessing the probability of infection. Debate also exists regarding the value of wound sampling, the types of wounds that should be sampled, and the sampling technique required to generate the most meaningful data. In the laboratory, consideration must be given to the relevance of culturing polymicrobial specimens, the value in identifying one or more microorganisms, and the microorganisms that should be assayed for antibiotic susceptibility. Although appropriate systemic antibiotics are essential for the treatment of deteriorating, clinically infected wounds, debate exists regarding the relevance and use of antibiotics (systemic or topical) and antiseptics (topical) in the treatment of nonhealing wounds that have no clinical signs of infection. In providing a detailed analysis of wound microbiology, together with current opinion and controversies regarding wound assessment and treatment, this review has attempted to capture and address microbiological aspects that are critical to the successful management of microorganisms in wounds.
Adjuvant Activity of Monophosphoryl Lipid A for Nasal and Oral Immunization with Soluble or Liposome-Associated Antigen
Authors: Childers Noel K. , Miller Keri L. , Tong Giang , Llarena Juan Carlos , Greenway Terrance , Ulrich J. Terry , Michalek Suzanne M.
Published: July 2002

The effectiveness of monophosphoryl lipid A (MPL) as a mucosal adjuvant was investigated following oral or intranasal (i.n.) administration of an aqueous adjuvant formulation of MPL (MPL-AF) added to soluble antigen or liposomal antigen or incorporated into liposomal antigen membranes. Groups of BALB/c female mice were immunized with 50 to 100 μg of free or liposomalStreptococcus mutanscrude glucosyltransferase (C-GTF) with or without MPL-AF added to the vaccine or incorporated into the liposomal membrane. Plasma, saliva, vaginal wash, and fecal extract samples were collected biweekly following immunization and assessed for antigen-specific antibody activity by enzyme-linked immunosorbent assay (ELISA). Mice immunized by the i.n. route had higher levels of salivary, plasma, and vaginal immunoglobulin A (IgA) anti-C-GTF responses and higher levels of plasma IgG anti-C-GTF than the orally immunized groups. A second administration of the vaccine 14 weeks after the initial immunization resulted in an anamnestic response to C-GTF resulting in 10- and 100-fold increases in saliva and plasma IgA and plasma IgG, respectively (in the i.n. immunized groups). Mice receiving a second i.n. immunization with liposomal antigen and MPL-AF had higher salivary IgA anti-C-GTF responses than mice immunized with antigen plus MPL-AF or liposomal antigen (P< 0.05). Plasma IgG anti-C-GTF activity was highest in mice immunized by the i.n. route with antigen formulations containing MPL-AF (P< 0.05). These results demonstrate the effectiveness of MPL-AF as an adjuvant for potentiating mucosal and systemic immune responses to liposomal C-GTF following i.n. immunization.
Comparison of Abilities of
Salmonella enterica
Serovar Typhimurium
aroA aroD
and
aroA htrA
Mutants To Act as Live Vectors
Authors: Roberts Mark , Chatfield Steven , Pickard Derek , Li Jingli , Bacon Andrew
Published: July 2002

We compared the ability of
Salmonella enterica
serovar Typhimurium SL1344
aroA aroD
(BRD509) and
aroA htrA
(BRD807) mutants to act as live vectors for delivery of fragment C of tetanus toxin (FrgC). FrgC was expressed in these strains from either pTETnir15 or pTEThtrA1. BRD509FrgC
+
strains elicited ∼2-log-higher serum anti-FrgC antibody titers than BRD807FrgC
+
strains. All mice immunized with BRD807pTEThtrA1, BRD509pTEThtrA1, and BRD509pTETnir15 (but not BRD807pTETnir15) were protected against tetanus.
Activation of Nuclear Factor κB and Induction of Inducible Nitric Oxide Synthase byUreaplasma urealyticumin Macrophages
Authors: Li Y.-H. , Yan Z.-Q. , Jensen J. Skov , Tullus K. , Brauner A.
Published: July 2002

Chronic lung disease (CLD) of prematurity is an inflammatory disease with a multifactorial etiology. The importance ofUreaplasma urealyticumin the development of CLD is debated, and steroids produce some improvement in neonates with this disease. In the present study, the capability ofU. urealyticumto stimulate rat alveolar macrophages to produce nitric oxide (NO), express inducible nitric oxide synthase (iNOS), and activate nuclear factor κB (NF-κB) in vitro was characterized. The effect of NO on the growth ofU. urealyticumwas also investigated. In addition, the impact of dexamethasone and budesonide on these processes was examined. We found thatU. urealyticumantigen (≥4 × 107color-changing units/ml) stimulated alveolar macrophages to produce NO in a dose- and time-dependent manner (P< 0.05). This effect was further enhanced by gamma interferon (100 IU/ml;P< 0.05) but was attenuated by budesonide and dexamethasone (10−4to 10−6M) (P< 0.05). The mRNA and protein levels of iNOS were also induced in response toU. urealyticumand inhibited by steroids.U. urealyticumantigen triggered NF-κB activation, a possible mechanism for the induced iNOS expression, which also was inhibited by steroids. NO induced byU. urealyticumcaused a sixfold reduction of its own growth after infection for 10 h. Our findings imply thatU. urealyticummay be an important factor in the development of CLD. The host defense response againstU. urealyticuminfection may also be influenced by NO. The down-regulatory effect of steroids on NF-κB activation, iNOS expression, and NO production might partly explain the beneficial effect of steroids in neonates with CLD.
Disruption of the Genes Encoding Antigen 85A and Antigen 85B of
Mycobacterium tuberculosis
H37Rv: Effect on Growth in Culture and in Macrophages
Authors: Armitige Lisa Y. , Jagannath Chinnaswamy , Wanger Audrey R. , Norris Steven J.
Published: July 2002

The mechanism of pathogenesis of
Mycobacterium tuberculosis
is thought to be multifactorial. Among the putative virulence factors is the antigen 85 (Ag85) complex. This family of exported fibronectin-binding proteins consists of members Ag85A, Ag85B, and Ag85C and is most prominently represented by 85A and 85B. These proteins have recently been shown to possess mycolyl transferase activity and likely play a role in cell wall synthesis. The purpose of this study was to generate strains of
M. tuberculosis
deficient in expression of the principal members of this complex in order to determine their role in the pathogenesis of
M. tuberculosis
. Constructs of
fbpA
and
fbpB
disrupted with the kanamycin resistance marker ΩKm and containing varying amounts of flanking gene and plasmid vector sequences were then introduced as linear fragments into H37Rv by electroporation. Southern blot and PCR analyses revealed disruption of the homologous gene locus in one
fbpA
::ΩKm transformant and one
fbpB
::ΩKm transformant. The
fbpA
::ΩKm mutant, LAa1, resulted from a double-crossover integration event, whereas the
fbpB
::ΩKm variant, LAb1, was the product of a single-crossover type event that resulted in insertion of both ΩKm and plasmid sequences. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis confirmed that expression of the disrupted gene was not detectable in the
fbpA
and
fbpB
mutants. Analysis of growth rates demonstrated that the
fbpB
mutant LAb1 grew at a rate similar to that of the wild-type parent in enriched and nutrient-poor laboratory media as well as in human (THP-1) and mouse (J774.1A) macrophage-like cell lines. The
fbpA
mutant LAa1 grew similarly to the parent H37Rv in enriched laboratory media but exhibited little or no growth in nutrient-poor media and macrophage-like cell lines. The targeted disruption of two genes encoding mycolyl transferase and fibronectin-binding activities in
M. tuberculosis
will permit the systematic determination of their roles in the physiology and pathogenesis of this organism.
Activated T Cells Induce Macrophages To Produce NO and ControlLeishmania majorin the Absence of Tumor Necrosis Factor Receptor p55
Authors: Nashleanas Michelle , Scott Phillip
Published: July 2002

The ability to activate macrophages in vitro for nitric oxide production and killing ofLeishmania majorparasites is dependent on tumor necrosis factor, althoughL. major-infected mice lacking the TNF receptor p55 (TNFRp55−/−mice) or both the TNFRp55 and TNFRp75 (TNFRp55p75−/−mice) are able to produce NO in vivo and eliminate the parasites. Here we report that activated T cells cocultured with macrophages results in TNFR-independent activation sufficient to control parasites and that both CD40/CD40L and LFA-1 contribute to T-cell-mediated macrophage activation. Thus, anti-CD3-stimulated T cells activated TNFR-deficient macrophages, while T cells from CD40L−/−mice were partially defective in triggering NO production by TNFRp55p75−/−macrophages. Moreover, in the presence of gamma interferon, anti-CD40 monoclonal antibody (MAb) activated TNFR-deficient macrophages. Finally, MAb blockade of LFA-1 completely inhibited macrophage NO production. Our data indicate that T cells can activate macrophages in the absence of TNF, thus providing a mechanism for how TNFR-deficient mice can control intracellular pathogens.
CXC Chemokine Receptor CXCR2 Is Essential for Protective Innate Host Response in MurinePseudomonas aeruginosaPneumonia
Authors: Tsai Wan C. , Strieter Robert M. , Mehrad Borna , Newstead Michael W. , Zeng Xianying , Standiford Theodore J.
Published: July 2002

Pulmonary infection due toPseudomonas aeruginosahas emerged as a leading cause of mortality. A vigorous host response is required to effectively clear the organisms from the lungs. This host defense is dependent on the recruitment and activation of neutrophils and macrophages. A family of chemotactic cytokines (chemokines) has been shown to participate in this protective response. In this study, we assessed the role of the ELR+(glutamic acid-leucine-arginine motif positive) CXC chemokines and their CXC chemokine receptor (CXCR2) in lung antibacterial host defense. The intratracheal administration ofPseudomonasto mice resulted in the time-dependent influx of neutrophils to the lung, peaking at 12 to 24 h after inoculation. The influx of neutrophils was associated with a similar time-dependent expression of the ELR+CXC chemokines, KC, macrophage inflammatory protein 2 (MIP-2), and lipopolysaccharide-induced CXC chemokine (LIX). Selective neutralization of MIP-2 or KC resulted in modest changes in neutrophil influx but no change in bacterial clearance or survival. However, neutralization of CXCR2 resulted in a striking increase in mortality, which was associated with a marked decrease in neutrophil recruitment and bacterial clearance. Conversely, the site-specific transgenic expression of KC resulted in enhanced clearance of bacteria afterPseudomonaschallenge. This study indicates that ELR+CXC chemokines are critical mediators of neutrophil-mediated host defense inPseudomonaspneumonia.
Cl
−
Secretion in Colonic Epithelial Cells Induced by the
Vibrio parahaemolyticus
Hemolytic Toxin Related to Thermostable Direct Hemolysin
Authors: Takahashi Akira , Kenjyo Noriko , Imura Kinichi , Myonsun Yoh , Honda Takeshi
Published: July 2002

A hemolytic toxin related to thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH), produced by Kanagawa-phenomenon-negative
Vibrio parahaemolyticus
is suspected of playing an important, but yet-to-be-elucidated role in diarrhea caused by this organism. In cultured human colonic epithelial cells, TRH increases Cl
−
secretion, followed by elevation of intracellular calcium.
Human T- and B-Cell Responses toSchistosoma mansoniRecombinant Glyceraldehyde 3-Phosphate Dehydrogenase Correlate with Resistance to Reinfection withS. mansoniorSchistosoma haematobiumafter Chemotherapy
Authors: El Ridi Rashika , Shoemaker Charles B. , Farouk Faten , El Sherif Naglaa H. , Afifi Ahmed
Published: July 2002

Recently we reported that human T- and B-cell recognition of a 42-kDa protein (p42) in soluble extracts of adultSchistosoma mansoniworms correlates with resistance to reinfection withS. mansoniorS. haematobium. Amino acid microsequencing of p42 revealed that it consists predominantly of schistosome glyceraldehyde 3-phosphate dehydrogenase (SG3PDH). We have expressed SG3PDH inEscherichia coliand purified the recombinant protein in a soluble and enzymatically active form. Recombinant SG3PDH (rSG3PDH) reacted with human monospecific antibodies to p42. Lymphoproliferation and production of interleukin-4 and gamma interferon (IFN-γ) after in vitro stimulation with rSG3PDH and serum isotype responses to rSG3PDH were examined in individuals with extremes of resistance and susceptibility to reinfection after treatment of previousS. mansoniorS. haematobiuminfection. Lymphoproliferation and IFN-γ production in response to rSG3PDH and the presence of serum immunoglobulin G1 (IgG1), IgG3, and IgA antibodies to rSG3PDH generally characterized individuals who are resistant to reinfection after chemotherapy. The data indicate that T- and B-cell immune reactivity to rSG3PDH correlates with resistance to reinfection, confirming previous studies identifying SG3PDH as a target of protective immunity in humans, and suggest that SG3PDH should be investigated as a possible vaccine for human schistosomiasis.
Legionella pneumophilaEntry GenertxAIs Involved in Virulence
Authors: Cirillo Suat L. G. , Bermudez Luiz E. , El-Etr Sahar H. , Duhamel Gerald E. , Cirillo Jeffrey D.
Published: July 2002

Successful parasitism of host cells by intracellular pathogens involves adherence, entry, survival, intracellular replication, and cell-to-cell spread. Our laboratory has been examining the role of early events, adherence and entry, in the pathogenesis of the facultative intracellular pathogenLegionella pneumophila. Currently, the mechanisms used byL. pneumophilato gain access to the intracellular environment are not well understood. We have recently isolated three loci, designatedenh1,enh2, andenh3, that are involved in the ability ofL. pneumophilato enter host cells. One of the genes present in theenh1locus,rtxA, is homologous to repeats in structural toxin genes (RTX) found in many bacterial pathogens. RTX proteins from other bacterial species are commonly cytotoxic, and some of them have been shown to bind to β2integrin receptors. In the current study, we demonstrate that theL. pneumophila rtxAgene is involved in adherence, cytotoxicity, and pore formation in addition to its role in entry. Furthermore, anrtxAmutant does not replicate as well as wild-typeL. pneumophilain monocytes and is less virulent in mice. Thus, we conclude that the entry genertxAis an important virulence determinant inL. pneumophilaand is likely to be critical for the production of Legionnaires' disease in humans.
EnteropathogenicEscherichia coliInfection Induces Expression of the Early Growth Response Factor by Activating Mitogen-Activated Protein Kinase Cascades in Epithelial Cells
Authors: de Grado Myriam , Rosenberger Carrie M. , Gauthier Annick , Vallance Bruce A. , Finlay B. Brett
Published: July 2002

EnteropathogenicEscherichia coli(EPEC) is an extracellular bacterial pathogen that infects the human intestinal epithelium and is a major cause of infantile diarrhea in developing countries. EPEC belongs to the group of attaching and effacing (A/E) pathogens. It uses a type III secretion system to deliver proteins into the host cell that mediate signal transduction events in host cells. We used gene array technology to study epithelial cell responses to EPEC infection at the level of gene expression. We found that EPEC induces the expression of several genes in infected HeLa cells by a lipopolysaccharide (LPS)-independent mechanism, including cytokines and early growth response factor 1 (Egr-1). The transcription factor Egr-1 is an immediate-early-induced gene that is activated in most cell types in response to stress. EPEC-induced upregulation ofegr-1is mediated by the activation of the MEK/extracellular signal-regulated kinase signal transduction pathway and is dependent on the type III secretion system.egr-1is also induced during infection of mice by the A/E pathogenCitrobacter rodentium, suggesting that both Egr-1 and the activation of this mitogen-activated protein kinase signal transduction pathway may play a role in disease.
Developmental Expression of Two Spore Wall Proteins during Maturation of the Microsporidian
Encephalitozoon intestinalis
Authors: Hayman J. Russell , Hayes Stanley F. , Amon Joseph , Nash Theodore E.
Published: July 2002

Microsporidia are intracellular eukaryotes that infect many animals and cause opportunistic infections in AIDS patients. The disease is transmitted via environmentally resistant spores. Two spore wall constituents from the microsporidian
Encephalitozoon intestinalis
were characterized. Spore wall protein 1 (SWP1), a 50-kDa glycoprotein recognized by monoclonal antibody (MAb) 11B2, was detected in developing sporonts and at low levels on the surfaces of mature spores. In contrast, SWP2, a 150-kDa glycoprotein recognized by MAb 7G7, was detected on fully formed sporonts and was more abundant on mature spores than SWP1. Nevertheless, the SWPs appeared to be complexed on the surfaces of mature spores. SWP1 and SWP2 are similar at the DNA and protein levels and have 10 conserved cysteines in the N-terminal domain, suggesting similar secondary structures. The C-terminal domain of SWP2 has a unique region containing 50 repeating 12- or 15-amino-acid units that lacks homology to known protein motifs. Antibodies from mice infected with
E. intestinalis
recognized SWP1 and SWP2. The characterization of two immunogenic SWPs from
E. intestinalis
will allow the study of exospore structure and function and may lead to the development of useful tools in the diagnosis and treatment of microsporidiosis.
DNA Vaccines Expressing a Fusion Product of Outer Surface Proteins A and C fromBorrelia burgdorferiInduce Protective Antibodies Suitable for Prophylaxis but Not for Resolution of Lyme Disease
Authors: Wallich Reinhard , Siebers Annette , Jahraus Oliver , Brenner Christiane , Stehle Thomas , Simon Markus M.
Published: July 2002

DNA vaccines encoding the outer surface protein A (OspA) ofBorrelia burgdorferihave been shown to induce protective humoral responses capable of preventing but not curing infection in mice. Subsequent studies showed that an established infection or disease could be resolved by passive transfer of antibodies to OspC. In the present study, DNA vaccines encoding either the OspC antigen alone or fused to OspA and under the transcriptional control of the human elongation factor 1α promoter were evaluated for their protective and/or curative potential. In contrast toospA-containing plasmids, none of the six constructs withospCalone were immunogenic in vivo, independent of whether they contained promoter or leader sequences fromospAand/orospC, or alternatively, the signal sequence of the human tissue plasminogen activator. Solely, a DNA vaccine encoding an OspA-OspC fusion product led to expression of the respective polypeptide chain in transfected cells in vitro and to the induction of OspA- and OspC-specific antibodies in vivo. Immune sera raised against the OspA-OspC fusion product conveyed full protection against subsequent infection, most probably via OspA-specific antibodies, but were unable to resolve infection.
Immunohistochemical Analysis of Cellular Infiltrate and Gamma Interferon, Interleukin-12, and Inducible Nitric Oxide Synthase Expression in Leprosy Type 1 (Reversal) Reactions before and during Prednisolone Treatment
Authors: Little David , Khanolkar-Young Saroj , Coulthart Anne , Suneetha Sujai , Lockwood Diana N. J.
Published: July 2002

The effects of prednisolone treatment on the cellularity and cytokine (gamma interferon, interleukin-12, and inducible nitric oxide synthase) profiles of leprosy skin type 1 (reversal) reactions were studied using immunohistochemistry. Skin biopsies were taken from 15 patients with leprosy type 1 (reversal) reactions at days 0, 7, 28, and 180 after the start of steroid treatment. Prednisolone treatment had little effect at day 7, but by day 28 significant decreases were found in cytokine levels. Some patients maintained cytokine production at days 28 and 180. These results illustrate the strong Th1 profile of type 1 reactional lesions, the slow response to steroid therapy, and continuing activity at 180 days.
Calprotectin Expression Inhibits Bacterial Binding to Mucosal Epithelial Cells
Authors: Nisapakultorn Kanokwan , Ross Karen F. , Herzberg Mark C.
Published: July 2002

Squamous mucosal epithelial cells constitutively express calprotectin in the cytoplasm. To study how this antimicrobial protein complex confers epithelial resistance to invading bacteria, an epithelial cell line was stably transfected to express the calprotectin complex. Cells expressing calprotectin resist invasion byListeria monocytogenesandSalmonella entericaserovar Typhimurium. Calprotectin expression was accompanied by altered actin organization, increased α3 integrin expression, and spreading cell morphology. In this study, we assessed whether calprotectin expression affects bacterial binding and uptake. Threefold-fewerListeriaorganisms bound to the surfaces of calprotectin-expressing cells, and 10-fold fewer were localized intracellularly by immunofluorescence. Similarly, fewerSalmonellaorganisms bound to cells expressing calprotectin. Calprotectin-expressing and sham-transfected cells showed similar levels of expression of surface E-cadherin and intracellular adhesion molecule 1 (ICAM-1) by flow cytometry. Calprotectin-expressing transfectants expressed calprotectin on the cell surface as well as in the cytosol. In conclusion, two bacterial pathogens showed reduced binding to calprotectin-expressing epithelial cells. Calprotectin-expressing cells appeared to have internalized disproportionately fewerListeriaorganisms, suggesting that reduced binding and translocation supplemented direct antimicrobial effects in calprotectin-expressing cells.
Expression and Functional Properties of the
Streptococcus intermedius
Surface Protein Antigen I/II
Authors: Petersen F.C. , Pasco S. , Ogier J. , Klein J. P. , Assev S. , Scheie A. A.
Published: July 2002

Streptococcus intermedius
is associated with deep-seated purulent infections. In this study, we investigated expression and functional activities of antigen I/II in
S. intermedius
. The
S. intermedius
antigen I/II appeared to be cell surface associated, with a molecular mass of approximately 160 kDa. Northern blotting indicated that the
S. intermedius
NCTC 11324 antigen I/II gene was transcribed as a monocistronic message. Maximum expression was seen during the early exponential phase. Insertional inactivation of the antigen I/II gene resulted in reduced hydrophobicity during early exponential phase, whereas no effect was detected during mid- and late exponential phases. Binding to human fibronectin and laminin was reduced in the isogenic mutant, whereas binding to human collagen types I and IV and to rat collagen type I was not significant for either the wild type or the mutant. Compared to the wild type, the capacity of the isogenic mutant to induce interleukin 8 (IL-8) release by THP-1 monocytic cells was significantly reduced. The results indicate that the
S. intermedius
antigen I/II is involved in adhesion to human receptors and in IL-8 induction.
Heterogeneity of WildLeishmania majorIsolates in Experimental Murine Pathogenicity and Specific Immune Response
Authors: Kébaı̈er C. , Louzir H. , Chenik M. , Salah A. Ben , Dellagi K.
Published: July 2002

Virulence variability was investigated by analyzing the experimental pathogenicity of 19Leishmania majorstrains in susceptible BALB/c mice. Twelve strains were isolated from Tunisian patients with zoonotic cutaneous leishmaniasis; seven strains were isolated in Syria (n= 1), Saudi Arabia (n= 2), Jordan (n= 2), or Israel (n= 2). BALB/c mice were injected in the hind footpad with 2 × 106amastigotes of the various isolates, and lesion progression was recorded weekly for 9 weeks. Interleukin-4 (IL-4) and gamma interferon (IFN-γ) production of lymph node mononuclear cells activated in vitro with parasite antigens were evaluated 5 weeks after infection. We show that disease progression induced by differentL. majorisolates was largely heterogeneous although reproducible results were obtained when using the same isolate. Interestingly, isolates from the Middle East induced a more severe disease than did the majority of Tunisian isolates. Strains with the highest virulence tend to generate more IL-4 and less IFN-γ in vitro at week 5 postinfection as well as higher levels of early IL-4 mRNA in the lymph node draining the inoculation site at 16 h postinfection. These results suggest thatL. majorisolates from the field may differ in virulence, which influences the course of the disease induced in mice and the type of immune response elicited by the infected host.
Salmonella enterica
Serovar Gallinarum Requires the
Salmonella
Pathogenicity Island 2 Type III Secretion System but Not the
Salmonella
Pathogenicity Island 1 Type III Secretion System for Virulence in Chickens
Authors: Jones Michael A. , Wigley Paul , Page Kerrie L. , Hulme Scott D. , Barrow Paul A.
Published: July 2002

Salmonella enterica
serovar Gallinarum is a host-specific serotype that causes the severe systemic disease fowl typhoid in domestic poultry and a narrow range of other avian species but rarely causes disease in mammalian hosts. Specificity of the disease is primarily at the level of the reticuloendothelial system, but few virulence factors have been described other than the requirement for an 85-kb virulence plasmid. In this work, by making functional mutations in the type III secretion systems (TTSS) encoded by
Salmonella
pathogenicity island 1 (SPI-1) and SPI-2, we investigated the role of these pathogenicity islands in interactions between
Salmonella
serovar Gallinarum and avian cells in vitro and the role of these pathogenicity islands in virulence in chickens. The SPI-1 mutant showed decreased invasiveness into avian cells in vitro but was unaffected in its ability to persist within chicken macrophages. In contrast the SPI-2 mutant was fully invasive in nonphagocytic cells but failed to persist in macrophages. In chicken infections the SPI-2 mutant was attenuated while the SPI-1 mutant showed full virulence. In oral infections the SPI-2 mutant was not observed in the spleen or liver, and following intravenous inoculation it was cleared rapidly from these sites. SPI-2 function is required by
Salmonella
serovar Gallinarum for virulence, primarily through promoting survival within macrophages allowing multiplication within the reticuloendothelial system, but this does not preclude the involvement of SPI-2 in uptake from the gut to the spleen and liver. SPI-1 appears to have little effect on virulence and survival of
Salmonella
serovar Gallinarum in the host.
Hydrolysis of Interleukin-12 byPorphyromonas gingivalisMajor Cysteine Proteinases May Affect Local Gamma Interferon Accumulation and the Th1 or Th2 T-Cell Phenotype in Periodontitis
Authors: Yun Peter L. W. , Decarlo Arthur A. , Collyer Charles , Hunter Neil
Published: July 2002

Porphyromonas gingivaliscysteine proteinases (gingipains) have been associated with virulence in destructive periodontitis, a disease process variously considered to represent an unregulated stimulation of either T helper type 1 (Th1)- or Th2-type cells. Critical in maintaining Th1 activity is the response of T lymphocytes to environmental interleukin 12 (IL-12) in the form of up-regulation of gamma interferon (IFN-γ) production. Here we demonstrate that in the presence or absence of serum, gingipains were able to hydrolyze IL-12 and reduce the IL-12-induced IFN-γ production from CD4+T cells. However, the induction of IL-12 receptors on T cells by gingipains did not correlate with the enhancement of IFN-γ production. The gingipains cleaved IL-12 within the COOH-terminal region of the p40 and p35 subunit chains, which leads to IL-12 inactivity, whereas IL-2 in these assays was not affected. Inactivation of IL-12 by the gingipains could disrupt the cytokine balance or favor Th2 activities in the progression of periodontitis.
Regulation of
Staphylococcus aureus
Capsular Polysaccharide Expression by
agr
and
sarA
Authors: Luong Thanh , Sau Subrata , Gomez Marisa , Lee Jean C. , Lee Chia Y.
Published: July 2002

This study addresses the regulation of
Staphylococcus aureus
type 8 capsular polysaccharide (CP8) expression by the global regulators
agr
and
sarA
. We analyzed CP8 production,
cap8
-specific mRNA synthesis, and
blaZ
reporter gene activities of the transcriptional and translational fusions in strain Becker and its
agr
,
sarA
, and
agr-sarA
isogenic mutants during different phases of bacterial growth. In the wild-type strain,
cap8
mRNA was undetectable until the mid-logarithmic phase of growth, whereas CP8 production was undetectable until 2 h later, at the onset of stationary phase. The delay most likely reflects the time needed for completing CP8 synthesis resulting from translation of
cap8
mRNA. The
agr
mutation caused drastic reductions in CP8 production and
cap8
gene transcription, suggesting that
agr
is a major positive regulator of CP8 expression. The results of gene fusion studies indicated that regulation by
agr
is exerted at the transcriptional level. In contrast, the
sarA
mutation caused only a slight reduction in
cap8
mRNA synthesis and reporter gene activities. By comparing CP8 production and
cap8
transcription, we observed that
sarA
affected CP8 production both trancriptionally and posttranslationally. We showed that
agr
was a major activator for
cap
gene expression not only in type 8 strain Becker but also in strains representing the four
agr
groups.
Expression of Indoleamine 2,3-Dioxygenase, Tryptophan Degradation, and Kynurenine Formation during In Vivo Infection withToxoplasma gondii: Induction by Endogenous Gamma Interferon and Requirement of Interferon Regulatory Factor 1
Authors: Silva Neide M. , Rodrigues Cibele V. , Santoro Marcelo M. , Reis Luiz F. L. , Alvarez-Leite Jacqueline I. , Gazzinelli Ricardo T.
Published: July 2002

The induction of indoleamine 2,3-dioxygenase (INDO) expression and the tryptophan (Trp)-kynurenine (Kyn) metabolic pathway during in vivo infection withToxoplasma gondiiwas investigated. Decreased levels of Trp and increased formation of Kyn were observed in the lungs, brain, and serum from mice infected withT. gondii. Maximal INDO mRNA expression and enzyme activity were detected in the lungs at 10 to 20 days postinfection. Further, the induction of INDO mRNA expression, Trp degradation and Kyn formation were completely absent in tissues from mice deficient in IFN-γ (IFN-γ−/−) or IFN regulatory factor -1 (IRF-1−/−). These findings indicate the important role of endogenous IFN-γ and IRF-1 in the in vivo induction of the Trp-Kyn metabolic pathway during acute infection withT. gondii. In contrast, expression of INDO mRNA and its activity was preserved in the tissues of TNF-receptor p55- or inducible nitric oxide synthase-deficient mice infected withT. gondii. Together with the results showing the extreme susceptibility of the IFN-γ−/−and the IRF-1−/−mice to infection withT. gondii, our results indicate a possible involvement of INDO and Trp degradation in host resistance to early infection with this parasite.