bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessing the Role of
Pseudomonas aeruginosa
Surface-Active Gene Expression in Hexadecane Biodegradation in Sand
Authors: Holden P. A. , LaMontagne M. G. , Bruce A. K. , Miller W. G. , Lindow S. E.
Published: July 2002

Low pollutant substrate bioavailability limits hydrocarbon biodegradation in soils. Bacterially produced surface-active compounds, such as rhamnolipid biosurfactant and the PA bioemulsifying protein produced by
Pseudomonas aeruginosa
, can improve bioavailability and biodegradation in liquid culture, but their production and roles in soils are unknown. In this study, we asked if the genes for surface-active compounds are expressed in unsaturated porous media contaminated with hexadecane. Furthermore, if expression does occur, is biodegradation enhanced? To detect expression of genes for surface-active compounds, we fused the
gfp
reporter gene either to the promoter region of
pra
, which encodes for the emulsifying PA protein, or to the promoter of the transcriptional activator
rhlR
. We assessed green fluorescent protein (GFP) production conferred by these gene fusions in
P. aeruginosa
PG201. GFP was produced in sand culture, indicating that the
rhlR
and
pra
genes are both transcribed in unsaturated porous media. Confocal laser scanning microscopy of liquid drops revealed that
gfp
expression was localized at the hexadecane-water interface. Wild-type PG201 and its mutants that are deficient in either PA protein, rhamnolipid synthesis, or both were studied to determine if the genetic potential to make surface-active compounds confers an advantage to
P. aeruginosa
biodegrading hexadecane in sand. Hexadecane depletion rates and carbon utilization efficiency in sand culture were the same for wild-type and mutant strains, i.e., whether PG201 was proficient or deficient in surfactant or emulsifier production. Environmental scanning electron microscopy revealed that colonization of sand grains was sparse, with cells in small monolayer clusters instead of multilayered biofilms. Our findings suggest that
P. aeruginosa
likely produces surface-active compounds in sand culture. However, the ability to produce surface-active compounds did not enhance biodegradation in sand culture because well-distributed cells and well-distributed hexadecane favored direct contact to hexadecane for most cells. In contrast, surface-active compounds enable bacteria in liquid culture to adhere to the hexadecane-water interface when they otherwise would not, and thus production of surface-active compounds is an advantage for hexadecane biodegradation in well-dispersed liquid systems.
Genetic Complementation of an Outer Membrane Cytochrome
omcB
Mutant of
Shewanella putrefaciens
MR-1 Requires
omcB
Plus Downstream DNA
Authors: Myers Judith M. , Myers Charles R.
Published: July 2002

Anaerobically grown cells of the metal-reducing bacterium
Shewanella putrefaciens
MR-1 contain multiple outer membrane (OM) cytochromes. A gene replacement mutant (strain OMCB1) lacking the OM cytochrome OmcB is markedly deficient in the reduction of MnO
2
and exhibits reduced rates of Fe(III) reduction. The levels of other OM cytochromes are also decreased in OMCB1. Complementation of OMCB1 with wild-type
omcB
did not restore any of these defects. However, a 21-kb genomic fragment from MR-1, which included
omcB
and 19 kb of downstream DNA, fully restored MnO
2
and Fe(III) reduction and the full complement of OM cytochromes to OMCB1. A 14.7-kb DNA fragment, including
omcB
and 12 kb of downstream DNA, provided only a modest increase in MnO
2
reduction and OM cytochrome content, but it fully restored Fe(III) citrate reduction and partially restored FeOOH reduction. While
omcB
mRNA was readily detected in this complement, the OmcB protein was not detected in any cellular compartment. The restoration of Fe(III) reduction despite the absence of OmcB suggests that OmcB itself is not required for Fe(III) reduction. Another OM cytochrome, OmcA, was mislocalized to the cytoplasmic membrane of OMCB1. Only the 21-kb genomic fragment was able to restore proper localization of OmcA to the OM. This 21-kb fragment does not contain
omcA
, but it does contain several open reading frames (ORFs) downstream from
omcB
. The most downstream of these ORFs (
altA
) encodes a putative AraC-like transcriptional regulator. However, a gene replacement mutant of
altA
resembled the wild type with respect to MnO
2
reduction, OM cytochrome content, and the localization of OmcA and OmcB to the OM. Since OMCB1 continues to express genes immediately downstream from
omcB
, the lack of expression of this downstream DNA does not explain its phenotype or the need for the large complementing fragment. The results suggest that the DNA downstream of
omcB
must be present in
cis
in order to restore Fe(III) reduction, MnO
2
reduction, OM cytochrome content, and the localization of OmcA and OmcB to the OM.
Dothistroma pini
, a Forest Pathogen, Contains Homologs of Aflatoxin Biosynthetic Pathway Genes
Authors: Bradshaw Rosie E. , Bhatnagar Deepak , Ganley Rebecca J. , Gillman Carmel J. , Monahan Brendon J. , Seconi Janet M.
Published: July 2002

Homologs of aflatoxin biosynthetic genes have been identified in the pine needle pathogen
Dothistroma pini. D. pini
produces dothistromin, a difuranoanthraquinone toxin with structural similarity to the aflatoxin precursor versicolorin B. Previous studies with purified dothistromin suggest a possible role for this toxin in pathogenicity. By using an aflatoxin gene as a hybridization probe, a genomic
D. pini
clone was identified that contained four
dot
genes with similarity to genes in aflatoxin and sterigmatocystin gene clusters with predicted activities of a ketoreductase (
dotA
), oxidase (
dotB
), major facilitator superfamily transporter (
dotC
), and thioesterase (
dotD
). A
D. pini dotA
mutant was made by targeted gene replacement and shown to be severely impaired in dothistromin production, confirming that
dotA
is involved in dothistromin biosynthesis. Accumulation of versicolorin A (a precursor of aflatoxin) by the
dotA
mutant confirms that the
dotA
gene product is involved in an aflatoxin-like biosynthetic pathway. Since toxin genes have been found to be clustered in fungi in every case analyzed so far, it is speculated that the four
dot
genes may comprise part of a dothistromin biosynthetic gene cluster. A fifth gene,
ddhA
, is not a homolog of aflatoxin genes and could be at one end of the dothistromin cluster. These genes will allow comparative biochemical and genetic studies of the aflatoxin and dothistromin biosynthetic pathways and may also lead to new ways to control
Dothistroma
needle blight.
Extensive Set of 16S rRNA-Based Probes for Detection of Bacteria in Human Feces
Authors: Harmsen Hermie J. M. , Raangs Gerwin C. , He Tao , Degener John E. , Welling Gjalt W.
Published: July 2002

For the detection of six groups of anaerobic bacteria in human feces, we designed seven new 16S rRNA-based oligonucleotide probes. This set of probes extends the current set of probes and gives more data on the composition of the human gut flora. Probes were designed for
Phascolarctobacterium
and relatives (Phasco741),
Veillonella
(Veil223),
Eubacterium hallii
and relatives (Ehal1469),
Lachnospira
and relatives (Lach571), and
Eubacterium cylindroides
and relatives (Ecyl387), and two probes were designed for
Ruminococcus
and relatives (Rbro730 and Rfla729). The hybridization conditions for the new probes were optimized for fluorescent in situ hybridization, and the probes were validated against a set of reference organisms. The probes were applied to fecal samples of 11 volunteers to enumerate their target bacterial groups. The Phasco741 and Veil223 probes both detected average numbers below 1% of the total number of bacteria as determined with the bacterial kingdom-specific Bact338 probe. The Ecyl387 probe detected about 1.4%, the Lach571 and Ehal1469 probes detected 3.8 and 3.6%, respectively, and a combination of the Rbro730 and Rfla729 probes detected 10.3%. A set of 15 probes consisting of probes previously described and those presented here were evaluated in hybridization with the fecal samples of the same volunteers. Together, the group-specific probes detected 90% of the total bacterial cells.
Vancomycin-Resistant
Enterococcus faecium
(VREF) from Norwegian Poultry Cluster with VREF from Poultry from the United Kingdom and The Netherlands in an Amplified Fragment Length Polymorphism Genogroup
Authors: Borgen Katrine , Wasteson Yngvild , Kruse Hilde , Willems Rob J. L.
Published: July 2002

The genetic relationship between 197 vancomycin-resistant
Enterococcus faecium
(VREF) isolates and 21 vancomycin-susceptible
E. faecium
isolates from Norwegian poultry was analyzed by amplified fragment length polymorphism (AFLP). The isolates were compared to 255 VREF isolates from various sources and countries. The Norwegian isolates constituted a relatively homogeneous population of
E. faecium
and clustered in a previously defined poultry AFLP genogroup.
Diagnosis of Tuberculosis Based on the Two Specific Antigens ESAT-6 and CFP10
Authors: van Pinxteren Laurens A. H. , Ravn Pernille , Agger Else Marie , Pollock John , Andersen Peter
Published: July 2002

Tests based on tuberculin purified protein derivative (PPD) cannot distinguish between tuberculosis infection,Mycobacterium bovisBCG vaccination, or exposure to environmental mycobacteria. The present study investigated the diagnostic potential of twoMycobacterium tuberculosis-specific antigens (ESAT-6 and CFP10) in experimental animals as well as during natural infection in humans and cattle. Both antigens were frequently recognized in vivo and in vitro based on the induction of delayed-type hypersensitivity responses and the ability to induce gamma interferon production by lymphocytes, respectively. The combination of ESAT-6 and CFP10 was found to be highly sensitive and specific for both in vivo and in vitro diagnosis. In humans, the combination had a high sensitivity (73%) and a much higher specificity (93%) than PPD (7%).
Induction of Hypergammaglobulinemia and Macrophage Activation by Silicone Gels and Oils in Female A.SW Mice
Authors: Naim J. O. , Satoh M. , Buehner N. A. , Ippolito K. M. L. , Yoshida H. , Nusz D. , Kurtelawicz L. , Cramer S. F. , Reeves W. H.
Published: July 2002

Although most published epidemiological studies have found little evidence of systemic autoimmune disease associated with silicone breast implants, there still remains a question of whether silicones can cause local and/or systemic immune dysfunction. This study further investigates the effects of silicones on autoantibody and immunoglobulin production and macrophage activation in female A.SW mice. Sixty mice were divided among four treatment groups receiving a 0.5-ml intraperitoneal injection of either phosphate-buffered saline (PBS), pristane, silicone gel, or silicone oil. Test bleeds were taken periodically for 6 months. In contrast to pristane, neither silicone gel nor silicone oil induced lupus-associated antinuclear autoantibodies (immunoglobulin G [IgG] anti-nRNP/Sm, Su, and ribosomal P) or lupus nephritis. However, serum IgM became elevated persistently within 1 month of silicone gel or silicone oil administration. Also, the level of IgG3 was clearly elevated in silicone oil-treated mice. In contrast, IgG1, IgG2a, and IgG2b levels were not affected greatly by either silicone gel or oil. Furthermore, peritoneal macrophages from silicone- and pristane-treated mice produced higher levels of interleukin-1β (IL-1β) and IL-6 than those from PBS-treated mice after lipopolysaccharide stimulation. These results suggest that silicone gels and oils are capable of inducing hypergammaglobulinemia and activating macrophages in female A.SW mice.
Earlier Detection of Human Immunodeficiency Virus Type 1 p24 Antigen and Immunoglobulin G and M Antibodies to p17 Antigen in Seroconversion Serum Panels by Immune Complex Transfer Enzyme Immunoassays
Authors: Hashida Seiichi , Ishikawa Setsuko , Hashinaka Kazuya , Nishikata Ichiro , Oka Shinichi , Ishikawa Eiji
Published: July 2002

For earlier diagnosis of human immunodeficiency virus type 1 (HIV-1) infection, the sensitivities of immune complex transfer enzyme immunoassays for HIV-1 p24 antigen and antibody immunoglobulin G (IgG) to HIV-1 p17 antigen were improved approximately 25- and 90-fold, respectively, over those of the previous immunoassays by performing solid-phase immunoreactions with shaking and increasing the serum sample volumes, and immune complex transfer enzyme immunoassay of antibody IgM to p17 antigen was also performed in the same way as the improved immunoassay of antibody IgG to p17 antigen. By the improved immunoassays, p24 antigen and antibody IgG to p17 antigen were detected earlier in 32 and 53%, respectively, of the HIV-1 seroconversion serum panels tested than before the improvements, and p24 antigen was detected as early as or earlier than HIV-1 RNA by reverse transcriptase-PCR (RT-PCR) in all of the panels tested. In 4 panels out of 19 tested, antibody IgG to p17 antigen or both antibodies IgG and IgM to p17 antigen were detected earlier than p24 antigen and RNA, although the antibody levels declined slightly before their steep increases usually observed after p24 antigen and RNA. Thus, the window period in diagnosis of HIV-1 infection can be shortened by detection of p24 antigen with the improved immunoassay as much as by detection of RNA with RT-PCR and, in some cases, more by detection of antibodies IgG and IgM to p17 antigen with the improved immunoassays than by detections of p24 antigen with the improved immunoassay and RNA with RT-PCR.
Pretreatment of Whole Blood for Use in Immunochromatographic Assays for Hepatitis B Virus Surface Antigen
Authors: Shin Hyeong-soon , Kim Chang-kyu , Shin Kwang-soon , Chung Hong-keun , Heo Tae-ryeon
Published: July 2002

Immunochromatographic assays (ICAs) are also referred to as rapid tests, since they are simple and the results can be obtained within minutes after manually loading a few drops of a sample into each sample well of the test device. However, whole blood cannot be tested with ICA kits due to the visual hindrance caused by the color of red blood cells (RBCs), unless a cell-removing device such as a filter is mounted on the kits. Thus, when testing with blood, the advantage of the ICA kit is lost because of the additional time and machines required to coagulate and separate whole blood before preparing the serum. To overcome this limitation, whole-blood samples were added to a pretreatment solution to decolor the RBCs; the resulting mixtures were then loaded into the sample wells of the test device. The pretreating solution was composed of hydrogen peroxide (H
2
O
2
) to decolor the RBCs, Sag 471 (Osi Specialties) to restrain the mixture from vigorous foaming, sodium azide (NaN
3
) to inhibit the enzyme, which generates excessive foam at the beginning of decolorization, and EDTA as a chelating agent. As a result of this pretreatment, whole blood could be used with the ICA kit without reducing its simplicity and rapidity.
Detection of Immunologically Significant Factors for Chronic Fatigue Syndrome Using Neural-Network Classifiers
Authors: Hanson S. J. , Gause W. , Natelson B.
Published: July 2002

Neural-network classifiers were used to detect immunological differences in groups of chronic fatigue syndrome (CFS) patients that heretofore had not shown significant differences from controls. In the past linear methods were unable to detect differences between CFS groups and non-CFS control groups in the nonveteran population. An examination of the cluster structure for 29 immunological factors revealed a complex, nonlinear decision surface. Multilayer neural networks showed an over 16% improvement in ann-fold resampling generalization test on unseen data. A sensitivity analysis of the network found differences between groups that are consistent with the hypothesis that CFS symptoms are a consequence of immune system dysregulation. Corresponding decreases in the CD19+B-cell compartment and the CD34+hematopoietic progenitor subpopulation were also detected by the neural network, consistent with the T-cell expansion. Of significant interest was the fact that, of all the cytokines evaluated, the only one to be in the final model was interleukin-4 (IL-4). Seeing an increase in IL-4 suggests a shift to a type 2 cytokine pattern. Such a shift has been hypothesized, but until now convincing evidence to support that hypothesis has been lacking.
Production ofBartonellaGenus-Specific Monoclonal Antibodies
Authors: Liang Zhongxing , La Scola Bernard , Lepidi Hubert , Raoult Didier
Published: July 2002

Monoclonal antibodies (MAbs) which react with heat-resistant proteins with molecular masses of 32 to 33 kDa of 14 differentBartonellaspecies were produced. These antibodies did not react with antigens of 26 diverse bacterial strains by microimmunofluorescence assay except MAb B3D4, which reacted withChlamydia psittaciandChlamydia trachomatisat low titers. The identification of a commonBartonellaantigenic protein will make it possible to later produce a diagnostic antigen by cloning and expressing it inEscherichia coli. Moreover, these MAbs allow allBartonellaspecies to be identified to the genus level.
Molecular Properties, Biology, and Clinical Implications of TT Virus, a Recently Identified Widespread Infectious Agent of Humans
Authors: Bendinelli Mauro , Pistello Mauro , Maggi Fabrizio , Fornai Claudia , Freer Giulia , Vatteroni Maria Linda
Published: July 2002

TT virus (TTV) was first described in 1997 by representational difference analysis of sera from non-A to non-G posttransfusion hepatitis patients and hence intensively investigated as a possible addition to the list of hepatitis-inducing viruses. The TTV genome is a covalently closed single-stranded DNA of approximately 3.8 kb with a number of characteristics typical of animal circoviruses, especially the chicken anemia virus. TTV is genetically highly heterogeneous, which has led investigators to group isolates into numerous genotypes and subtypes and has limited the sensitivity of many PCR assays used for virus detection. The most remarkable feature of TTV is the extraordinarily high prevalence of chronic viremia in apparently healthy people, up to nearly 100% in some countries. The original hypothesis that it might be an important cause of cryptogenic hepatitis has not been borne out, although the possibility that it may produce liver damage under specific circumstances has not been excluded. The virus has not yet been etiologically linked to any other human disease. Thus, TTV should be considered an orphan virus.
Chlamydia pneumoniae
and Atherosclerosis: Critical Assessment of Diagnostic Methods and Relevance to Treatment Studies
Authors: Boman Jens , Hammerschlag Margaret R.
Published: July 2002

A number of studies have found that inflammation of the vessel wall plays an essential role in both the initiation and progression of atherosclerosis and erosion and fissure and the eventual rupture of plaques. Chlamydia pneumoniae is one of the infectious agents that have been investigated as possible causes of this inflammation. Initial studies of the association of C. pneumoniae and cardiovascular disease (CVD) were seroepidemiologic, and these were followed by studies in which the organism was identified in vascular tissue from patients with CVD by electron microscopy, PCR and immunocytochemical staining (ICC). C. pneumoniae has also been isolated by culture from vascular tissue in a small number patients. However, no single serologic, PCR, or ICC assay has been used consistently across all studies. The assays used are also not standardized. Recent studies of serologic and PCR assays for diagnosis of C. pneumoniae infection have suggested that there may be substantial interlaboratory variation in the performance of these tests. It now appears that some of the inconsistency of results from study to study may be due, in part, to lack of standardized methods. Although initial seroepi-demiologic studies demonstrated a significantly increased risk of adverse cardiac outcome in patients who were seropositive, subsequent prospective studies found either small or no in-creased risk. In addition to the lack of consistent serologic criteria, recent evaluations have demonstrated inherent problems with performance of the most widely used serologic methods. Most importantly, we do not have a reliable serologic marker for chronic or persistent C. pneumoniae infection.
Clostridium perfringens
Beta-Toxin Forms Potential-Dependent, Cation-Selective Channels in Lipid Bilayers
Authors: Shatursky Oleg , Bayles Robert , Rogers Marianne , Jost B. Helen , Songer J. Glenn , Tweten Rodney K.
Published: July 2002

Recombinant beta-toxin from
Clostridium perfringens
type C was found to increase the conductance of bilayer lipid membranes (BLMs) by inducing channel activity. The channels exhibited a distribution of conductances within the range of 10 to 380 pS, with the majority of the channels falling into two categories of conductance at 110 and 60 pS. The radii of beta-toxin pores found for the conductance states of 110 and 60 pS were 12.7 and 11.1 Å, respectively. The single channels and the steady-state currents induced by beta-toxin across the BLMs exhibited ideal monovalent cation selectivity. Addition of divalent cations (Zn
2+
, Cd
2+
, or Mg
2+
) at a concentration of 2 mM increased the rate of beta-toxin insertion into BLMs and the single-channel conductance, while application of 5 mM Zn
2+
to a beta-toxin-induced steady-state current decreased the inward current by approximately 45%. The mutation of arginine 212 of beta-toxin to aspartate, previously shown to increase the 50% lethal dose of beta-toxin for mice nearly 13-fold, significantly reduced the ability of beta-toxin to form channels. These data support the hypothesis that the lethal action of beta-toxin is based on the formation of cation-selective pores in susceptible cells.
Salmonella entericaSerovar Typhimurium-Dependent Regulation of Inducible Nitric Oxide Synthase Expression in Macrophages by Invasins SipB, SipC, and SipD and Effector SopE2
Authors: Cherayil Bobby J. , McCormick Beth A. , Bosley Jacob
Published: July 2002

WhenSalmonella entericainvades mammalian cells, it activates signals leading to increased expression of inflammatory mediators. One such mediator is nitric oxide (NO), which is produced under control of the enzyme inducible NO synthase (iNOS). Induction of iNOS in response toSalmonellainfection has been demonstrated, but the bacterial effector molecules that regulate expression of the enzyme have not been identified. In the study reported here, an analysis ofSalmonella-dependent iNOS expression in macrophages was carried out. Wild-typeSalmonellastrains increased the levels of both iNOS protein and mRNA in murine macrophage cell lines in an invasion-independent fashion. Mutant strains lacking a functional pathogenicity island 1-encoded type III secretion system, as well as strains lacking the invasins SipB, SipC, and SipD, were impaired in iNOS induction. Complementation experiments indicated that all three of the invasins were required for induction of iNOS expression. These results suggested that an effector protein, translocated into macrophages via the type III secretion system in a SipB-, SipC-, and SipD-dependent manner, might be the ultimate mediator of iNOS induction. In keeping with this idea, a mutant strain deficient in SopE2, a recently described homolog of SopE, was found to be impaired in the induction of iNOS expression. These observations suggest that iNOS expression is regulated by signals activated by SopE2 (and possibly SopE) and that the role of SipB, SipC, and SipD in this process is to facilitate translocation of the relevant effector.
Identification of Vaccine Candidates for Experimental Visceral Leishmaniasis by Immunization with Sequential Fractions of a cDNA Expression Library
Authors: Melby Peter C. , Ogden Gary B. , Flores Hector A. , Zhao Weiguo , Geldmacher Christopher , Biediger Natalie M. , Ahuja Sunil K. , Uranga Jose , Melendez Maria
Published: July 2002

Visceral leishmaniasis caused by the intracellular parasiteLeishmania donovaniis a significant public health problem in many regions of the world. Because of its large genome and complex biology, developing a vaccine for this pathogen has proved to be a challenging task and, to date, protective recombinant vaccine candidates have not been identified. To tackle this difficult problem, we adopted a reductionist approach with the intention of identifying cDNA sequences in anL. donovaniamastigote cDNA library that collectively or singly conferred protection against parasite challenge in a murine model of visceral leishmaniasis. We immunized BALB/c mice with plasmid DNA isolated and pooled from 15 cDNA sublibraries (∼2,000 cDNAs/sublibrary). Following systemic challenge withL. donovani, mice immunized with 6 of these 15 sublibraries showed a significantly reduced (35- to 1,000-fold) hepatic parasite burden. Because of the complexity and magnitude of the sequential fractionation-immunization-challenge approach, we restricted our attention to the two sublibraries that conferred the greatest in vivo protection. From one of these two sublibraries, we identified several groups of cDNAs that afforded protection, including a set of nine novel cDNAs and, surprisingly, a group of five cDNAs that encodedL. donovanihistone proteins. At each fractionation step, the cDNA sublibraries or the smaller DNA fractions that afforded in vivo protection against the parasite also induced in vitro parasite-specific T helper 1 immune responses. Our studies demonstrate that immunization with sequential fractions of a cDNA library is a powerful strategy for identifying anti-infective vaccine candidates.
Tumor Necrosis Factor Alpha p55 Receptor Is Important for Development of Memory Responses to Blood-Stage Malaria Infection
Authors: Li Ching , Langhorne Jean
Published: July 2002

Tumor necrosis factor alpha (TNF-α) is associated with malarial pathology in both humans and mice. InPlasmodium chabaudi chabaudi(AS) infections, the production of TNF-α and reactive metabolites from macrophages are also thought to play a role in controlling acute parasitemia. Since many of the biological functions of TNF-α are effected through the p55 receptor (p55R), mice made defective in this receptor via a targeted gene disruption (p55R−/−) have been used to study its involvement in the immune response againstP. chabaudi chabaudiand in the pathology associated with this infection. In the absence of the p55R, mice could overcome their primary infection, although higher acute-blood-stage parasitemias and more significant recrudescences were observed. Hypoglycemia, hypothermia, loss of erythrocytes, and loss of body weight, which occur transiently in this infection, were exacerbated by the lack of the p55R, but the differences were small, suggesting that other factors affect these symptoms. In contrast to wild-type (WT) mice, a second challenge infection in p55R−/−mice resulted in a course of infection similar to a primary infection. The malaria-specific immunoglobulin G antibody response of p55R−/−mice was lower than that of WT mice and was not increased by the second challenge infection. These data suggest that p55R−/−mice do not develop an efficient memory B-cell response against malarial infection and that this antibody response is important in immunity to reinfection.
Cellular and Cytokine Correlates of Mucosal Protection in Murine Model of Oral Candidiasis
Authors: Elahi Shokrollah , Pang Gerald , Clancy Robert , Ashman Robert B.
Published: July 2002

Host protection againstCandida albicansinfection in a model of oral candidiasis involving infection-prone [DBA/2 (H-2d)] and less infection-prone [BALB/c (H-2d)] mouse strains was analyzed in terms of antibody and cellular responses, and in terms of cytokine patterns from regional lymph node cells. There was a selective expansion of γ/δ+T-cell receptor cells, which correlated with the patterns of colonization in both mouse strains, with higher numbers of γ/δ T cells detected in BALB/c mice. Antigen-induced T-cell proliferation was significantly higher in BALB/c mice than in DBA/2 mice. Higher levels of serum immunoglobulin G (IgG) and salivary IgA antibodies were detected in BALB/c mice than in DBA/2 mice, but only after the infection was cleared. The cervical lymph node cells from infected mice were assessed for interleukin-4 (IL-4), IL-12, and gamma interferon (IFN-γ) mRNA gene expression by reverse transcription-PCR and protein production in the culture supernatants following restimulation in vitro. In BALB/c mice, an early increase in levels of IL-4, IFN-γ, and IL-12 correlated with rapid elimination ofC. albicans. In DBA/2 mice, where resolution of infection was delayed, IL-4 message expression was delayed and the IL-4 secretion level was lower. Neutralization of IL-4 by multiple injections of an anti-IL-4 monoclonal antibody in BALB/c mice resulted in increased carriage rate and delayed clearance of the yeasts. Collectively, the data suggest that the T-cell response toC. albicansin the regional lymph nodes which correlates best with rapid oral clearance ofC. albicansis a balanced Th0 cytokine response involving early secretion of both IFN-γ and IL-4.
Integrated Genomic Map from Uropathogenic
Escherichia coli
J96
Authors: Melkerson-Watson Lyla J. , Rode Christopher K. , Zhang Lixin , Foxman Betsy , Bloch Craig A.
Published: July 2002

Escherichia coli
J96 is a uropathogen having both broad similarities to and striking differences from nonpathogenic, laboratory
E. coli
K-12. Strain J96 contains three large (>100-kb) unique genomic segments integrated on the chromosome; two are recognized as pathogenicity islands containing urovirulence genes. Additionally, the strain possesses a fourth smaller accessory segment of 28 kb and two deletions relative to strain K-12. We report an integrated physical and genetic map of the 5,120-kb J96 genome. The chromosome contains 26
Not
I, 13
Bln
I, and 7 I-
Ceu
I macrorestriction sites. Macrorestriction mapping was rapidly accomplished by a novel transposon-based procedure: analysis of modified minitransposon insertions served to align the overlapping macrorestriction fragments generated by three different enzymes (each sharing a common cleavage site within the insert), thus integrating the three different digestion patterns and ordering the fragments. The resulting map, generated from a total of 54 mini-Tn
10
insertions, was supplemented with auxanography and Southern analysis to indicate the positions of insertionally disrupted aminosynthetic genes and cloned virulence genes, respectively. Thus, it contains not only physical, macrorestriction landmarks but also the loci for eight housekeeping genes shared with strain K-12 and eight acknowledged urovirulence genes; the latter confirmed clustering of virulence genes at the large unique accessory chromosomal segments. The 115-kb J96 plasmid was resolved by pulsed-field gel electrophoresis in
Not
I digests. However, because the plasmid lacks restriction sites for the enzymes
Bln
I and I-
Ceu
I, it was visualized in
Bln
I and I-
Ceu
I digests only of derivatives carrying plasmid inserts artificially introducing these sites. Owing to an I-
Sce
I site on the transposon, the plasmid could also be visualized and sized from plasmid insertion mutants after digestion with this enzyme. The insertional strains generated in construction of the integrated genomic map provide useful physical and genetic markers for further characterization of the J96 genome.
Maturation of Human Dendritic Cells by Cell Wall Skeleton ofMycobacterium bovisBacillus Calmette-Guérin: Involvement of Toll-Like Receptors
Authors: Tsuji Shoutaro , Matsumoto Misako , Takeuchi Osamu , Akira Shizuo , Azuma Ichiro , Hayashi Akira , Toyoshima Kumao , Seya Tsukasa
Published: July 2002

The constituents of mycobacteria are an effective immune adjuvant, as observed with complete Freund's adjuvant. In this study, we demonstrated that the cell wall skeleton ofMycobacterium bovisbacillus Calmette-Guérin (BCG-CWS), a purified noninfectious material consisting of peptidoglycan, arabinogalactan, and mycolic acids, induces maturation of human dendritic cells (DC). Surface expression of CD40, CD80, CD83, and CD86 was increased by BCG-CWS on human immature DC, and the effect was similar to those of interleukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), heat-killed BCG, and viable BCG. BCG-CWS induced the secretion of TNF-α, IL-6, and IL-12 p40. CD83 expression was increased by a soluble factor secreted from BCG-CWS-treated DC and was completely inhibited by monoclonal antibodies against TNF-α. BCG-CWS-treated DC stimulated extensive allogeneic mixed lymphocyte reactions. The level of TNF-α secreted through BCG-CWS was partially suppressed in murine macrophages with no Toll-like receptor 2 (TLR 2) or TLR4 and was completely lost in TLR2 and TLR4 double-deficient macrophages. These results suggest that the BCG-CWS induces TNF-α secretion from DC via TLR2 and TLR4 and that the secreted TNF-α induces the maturation of DC per se.
Immunogenicity of the B Monomer ofEscherichia coliHeat-Labile Toxin Expressed on the Surface ofStreptococcus gordonii
Authors: Ricci Susanna , Medaglini Donata , Rush Catherine M. , Marcello Alessandro , Peppoloni Samuele , Manganelli Riccardo , Palú Giorgio , Pozzi Gianni
Published: July 2002

The B monomer of theEscherichia coliheat-labile toxin (LTB) was expressed on the surface of the human oral commensal bacteriumStreptococcus gordonii. Recombinant bacteria expressing LTB were used to immunize BALB/c mice subcutaneously and intragastrically. The LTB monomer expressed on the streptococcal surface proved to be highly immunogenic, as LTB-specific immunoglobulin G (IgG) serum titers of 140,000 were induced after systemic immunization. Most significantly, these antibodies were capable of neutralizing the enterotoxin in a cell neutralization assay. Following mucosal delivery, antigen-specific IgA antibodies were found in feces and antigen-specific IgG antibodies were found in sera. Analysis of serum IgG subclasses showed a clear predominance of IgG1 when recombinant bacteria were inoculated subcutaneously, while a prevalence of IgG2a was observed upon intragastric delivery, suggesting, in this case, the recruitment of a Th1 type of immune response.
Acquisition of Coinfection and Simultaneous Transmission of
Borrelia burgdorferi
and
Ehrlichia phagocytophila
by
Ixodes scapularis
Ticks
Authors: Levin Michael L. , Fish Durland
Published: July 2002

The agents of Lyme disease (
Borrelia burgdorferi
) and human granulocytic ehrlichiosis (
Ehrlichia phagocytophila
) are both transmitted by the tick
Ixodes scapularis
. In nature, ticks are often infected with both agents simultaneously. We studied whether previous infection with either
Borrelia
or
Ehrlichia
in ticks would affect acquisition and transmission of a second pathogen.
Ehrlichia
-infected
I. scapularis
nymphs were fed upon
Borrelia
-infected mice, and
Borrelia
-infected
I. scapularis
nymphs were fed upon
Ehrlichia
-infected mice. The efficiency with which previously infected nymphal ticks acquired a second pathogen from infected hosts was compared to that of uninfected ticks. An average of 51% ± 15% of ticks acquired
Ehrlichia
from infected mice regardless of their prior infection status with
Borrelia
. An average of 85% ± 10% of ticks acquired
Borrelia
from infected mice regardless of their prior infection status with
Ehrlichia
. Also, we assessed the efficiency with which individual nymphs could transmit either agent alone, or both agents simultaneously, to individual susceptible hosts. An average of 76% ± 9% of
Borrelia
-infected ticks and 84% ± 10% of
Ehrlichia
-infected ticks transmitted these agents to mice regardless of the presence of the other pathogen. There was no evidence of interaction between the agents of Lyme disease and human granulocytic ehrlichiosis in
I. scapularis
ticks. The presence of either agent in the ticks did not affect acquisition of the other agent from an infected host. Transmission of the agents of Lyme disease and human granulocytic ehrlichiosis by individual ticks was equally efficient and independent. Dually infected ticks transmitted each pathogen to susceptible hosts as efficiently as ticks infected with only one pathogen.
Identification and Expression of a
Mycoplasma gallisepticum
Surface Antigen Recognized by a Monoclonal Antibody Capable of Inhibiting Both Growth and Metabolism
Authors: Yoshida Shigeto , Fujisawa Ayumi , Tsuzaki Yoshinari , Saitoh Shuji
Published: July 2002

In order to identify antigenic proteins of
Mycoplasma gallisepticum
, monoclonal antibodies (MAbs) against virulent
M. gallisepticum
R strain were produced in mice. MAb 35A6 was selected for its abilities to inhibit both growth and metabolism of
M. gallisepticum
in vitro. The MAb recognized a membrane protein with an apparent molecular mass of 120 kDa. The corresponding gene, designated the mgc3 gene, was cloned from an
M. gallisepticum
genomic DNA expression library and sequenced. The mgc3 gene is a homologue of the ORF6 gene encoding 130-kDa protein in the P1 operon of
M. pneumoniae
and is localized downstream of the mgc1 gene, a homologue of the P1 gene. To assess the characteristics of MGC3 protein, all 10 TGA codons in the mgc3 gene, which encode a tryptophan in the
Mycoplasma
species, were replaced with TGG codons, and recombinant fowlpox viruses (FPV) harboring the altered mgc3 gene were constructed. One of the recombinant FPVs was improved to express MGC3 protein on the cell surface in which the signal peptide of MGC3 protein was replaced with one from Marek's disease virus gB. These results should provide the impetus to develop a vaccine based on MGC3 protein which can induce antibodies with both growth inhibition and metabolic-inhibition activities using a recombinant FPV.
Local Anticandidal Immune Responses in a Rat Model of Vaginal Infection by and Protection againstCandida albicans
Authors: de Bernardis Flavia , Santoni Giorgio , Boccanera Maria , Spreghini Elisabetta , Adriani Daniela , Morelli Luisella , Cassone Antonio
Published: July 2002

Humoral (antibody [Ab]) and cellularCandida-specific immune responses in the vaginas of pseudoestrus rats were investigated during three successive infections byCandida albicans. After the first, protective infection, Abs against mannan and aspartyl proteinase antigens were present in the vaginal fluid, and their titers clearly increased during the two subsequent, rapidly healing infections. In all animals, about 65 and 10% of vaginal lymphocytes (VL) were CD3+(T cells) and CD3−CD5+(B cells), respectively. Two-thirds of the CD3+T cells expressed the α/β and one-third expressed the γ/δ T-cell receptor (TCR). This proportion slightly fluctuated during the three rounds ofC. albicansinfection, but no significant differences between infected and noninfected rats were found. More relevant were the changes in the CD4+/CD8+T-cell ratio, particularly for cells bearing the CD25 (interleukin-2 receptor α) marker. In fact, a progressively increased number of both CD4+α/β TCR and CD4+CD25+VL was observed after the second and thirdCandidachallenges, reversing the high initial CD8+cell number of controls (estrogenized but uninfected rats). The CD3−CD5+cells also almost doubled from the first to the third infection. Analysis of the cytokines secreted in the vaginal fluid ofCandida-infected rats showed high levels of interleukin 12 (IL-12) during the first infection, followed by progressively increasing amounts of IL-2 and gamma interferon during the subsequent infections. No IL-4 or IL-5 was ever detected. During the third infection, VL with in vitro proliferative activity in response to an immunodominant mannoprotein antigen ofC. albicanswere present in the vaginal tissue. No response to this antigen by mitogen-responsive blood, lymph node, and spleen cells was found. In summary, the presence of protective Ab and T helper type 1 cytokines in the vaginal fluids, the in vitro proliferation of vaginal lymphocytes in response toCandidaantigenic stimulation, and the increased number of activated CD4+cells and some special B lymphocytes afterC. albicanschallenge constitute good evidence for induction of locally expressedCandida-specific Ab and cellular responses which are potentially involved in anticandidal protection at the vaginal level.
The SA85-1.1 Protein of theTrypanosoma cruzi trans-Sialidase Superfamily Is a Dominant T-Cell Antigen
Authors: Millar Amanda E. , Kahn Stuart J.
Published: July 2002

Trypanosoma cruzicurrently infects 18 million people, and 30% of those infected develop a chronic inflammatory process that causes significant morbidity or mortality. The major histocompatibility complex class II (MHC-II)-restricted T-cell response is critical to the control of the infection and to the ensuing inflammatory pathology. The specific epitopes or major antigens of this response have not been identified. The parasite simultaneously expresses variant members of thetrans-sialidase superfamily. To begin to analyze the MHC-II response to these variant proteins, the response to a single surface protein, SA85-1.1, was initiated. These studies have demonstrated that a biased gamma interferon (IFN-γ) response to the SA85-1.1 protein develops duringT. cruziinfection. In addition, adoptive transfer of a CD4 clone that recognizes an SA85-1.1 epitope, named epitope 1, and immunization with a peptide encoding epitope 1 were protective and suggested that epitope 1 may be immunodominant. In this report IFN-γ intracellular staining demonstrated that splenocytes from acutely and chronically infected mice, incubated with SA85-1.1 protein or peptides that encode epitope 1, result in IFN-γ synthesis by 4 to 6% of the splenic CD4 cells. These data indicate that duringT. cruziinfection epitope 1 is a major epitope and that 4 to 6% of the CD4 cells are stimulated by a singletrans-sialidase superfamily epitope and suggest that a combination oftrans-sialidase superfamily proteins combines to stimulate a majority of CD4 cells. These data suggest that duringT. cruziinfection the CD4 response to thetrans-sialidase superfamily is critical to the protective response and to the ensuing chronic inflammatory pathology.
Lack of Protection in Mice and Necrotizing Bronchointerstitial Pneumonia with Bronchiolitis in Guinea Pigs Immunized with Vaccines Directed against the hsp60 Molecule ofMycobacterium tuberculosis
Authors: Turner Oliver C. , Roberts Alan D. , Frank Anthony A. , Phalen Susan W. , McMurray David M. , Content Jean , Denis Olivier , D'Souza Sushila , Tanghe Audrey , Huygen Kris , Orme Ian M.
Published: July 2002

In this study, the hsp60 and hsp70 heat shock protein antigens ofMycobacterium tuberculosiswere tested as potential vaccine candidates, using purified recombinant protein antigens or antigens encoded in the form of a DNA plasmid vaccine. Guinea pigs vaccinated with a mixture of the two proteins showed no evidence of resistance to low-dose aerosol challenge infection and quickly developed severe lung damage characterized by necrotizing bronchointerstitial pneumonia and bronchiolitis. As a result, we turned instead to a DNA vaccination approach using a plasmid encoding the hsp60 antigen ofM. tuberculosis. Although immunogenic in mice, vaccination with plasmid DNA encoding hsp60 was not protective in that model or in the guinea pig model and again gave rise to similar severe lung damage. This study seriously questions the safety of vaccines against tuberculosis that target highly conserved heat shock proteins.
Tuberculosis DNA Vaccine Encoding Ag85A Is Immunogenic and Protective When Administered by Intramuscular Needle Injection but Not by Epidermal Gene Gun Bombardment
Authors: Tanghe Audrey , Denis Olivier , Lambrecht Bénédicte , Motte Vinciane , van den Berg Thierry , Huygen Kris
Published: July 2002

Immunogenicity and protective efficacy of a DNA vaccine encoding Ag85A fromMycobacterium tuberculosiswere compared in BALB/c and C57BL (B6 and B10) mice immunized by intramuscular (i.m.) needle injection or epidermal gene gun (gg) bombardment. In BALB/c mice, gg immunization could induce elevated antibody and cytotoxic T lymphocyte responses with plasmid doses 50-fold lower than those required for i.m. immunization. Interleukin-2 (IL-2) and gamma interferon (IFN-γ) secretion, however, was much lower in gg-immunized than in i.m.-immunized BALB/c mice. On the other hand, C57BL mice reacted only very weakly to gg immunization, whereas elevated Ag85A-specific antibody, IL-2, and IFN-γ responses (significantly higher than in BALB/c mice) were detected following vaccination by the i.m. route. Antibody isotypes were indicative of Th2 activation following gg injection of BALB/c and of Th1 activation following i.m. injection of C57BL mice. Finally, C57BL but not BALB/c mice were protected by i.m. Ag85A DNA immunization against intravenousM. tuberculosischallenge, as measured by reduced numbers of CFU in spleen and lungs, compared to animals vaccinated with control DNA. Gene gun immunization was not effective in either BALB/c or C57BL mice. These results indicate that i.m. DNA vaccination is the method of choice for the induction of protective Th1 type immune responses with the Ag85A tuberculosis DNA vaccine.
Identification and HLA Restriction of Naturally Derived Th1-Cell Epitopes from the SecretedMycobacterium tuberculosisAntigen 85B Recognized by Antigen-Specific Human CD4+T-Cell Lines
Authors: Mustafa Abu S. , Shaban Fatema A. , Abal Adnan T. , Al-Attiyah Raja , Wiker Harald G. , Lundin Knut E. A. , Oftung Fredrik , Huygen Kris
Published: July 2002

Antigen 85B (Ag85B/MPT59) is a major secreted protein fromMycobacterium tuberculosiswhich is a promising candidate antigen for inclusion in novel subunit vaccines against tuberculosis (TB). The present study was undertaken to map naturally derived T-cell epitopes fromM. tuberculosisAg85B in relation to major histocompatibility complex (MHC) class II restriction. Antigen-specific CD4+T-cell lines were established from HLA-typed TB patients andMycobacterium bovisBCG vaccinees by stimulation of peripheral blood mononuclear cells with purified Ag85B in vitro. The established T-cell lines were then tested for proliferation and gamma interferon (IFN-γ) secretion in response to 31 overlapping synthetic peptides (18-mers) covering the entire sequence of the mature protein. The results showed that the epitopes recognized by T-cell lines from TB patients were scattered throughout the Ag85B sequence whereas the epitopes recognized by T-cell lines from BCG vaccinees were located toward the N-terminal part of the antigen. The T-cell epitopes represented by peptides p2 (amino acids [aa] 10 to 27), p3 (aa 19 to 36), and p11 (aa 91 to 108) were frequently recognized by antigen-specific T-cell lines from BCG vaccinees in both proliferation and IFN-γ assays. MHC restriction analysis demonstrated that individual T-cell lines specifically recognized the complete Ag85B either in association with one of the selfHLA-DRB1,DRB3, orDRB4gene products or nonspecifically in a promiscuous manner. At the epitope level, panel studies showed that peptides p2, p3, and p11 were presented to T cells by HLA-DR-matched as well as mismatched allogeneic antigen-presenting cells, thus representing promiscuous epitopes. The identification of naturally derived peptide epitopes from theM. tuberculosisAg85B presented to Th1 cells in the context of multiple HLA-DR molecules strongly supports the relevance of this antigen to subunit vaccine design.