bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin-6 Deficiency Increases Inflammatory Bone Destruction
Authors: Balto Khaled , Sasaki Hajime , Stashenko Philip
Published: July 2002

Periapical bone destruction occurs as a consequence of pulpal infection. In previous studies, we showed that interleukin-1 (IL-1) is the primary stimulator of bone destruction in this model. IL-6 is a pleiotropic cytokine that is induced in these infections and has both pro- and anti-inflammatory activities. In the present study, we determined the role of IL-6 in regulating IL-1 expression and bone resorption. The first molars of IL-6 knockouts (IL-6−/−) and wild-type mice were subjected to surgical pulp exposure and infection with a mixture of four common pulpal pathogens, includingPrevotella intermedia,Fusobacterium nucleatum,Peptostreptococcus micros, andStreptococcus intermedius. Mice were killed after 21 days, and bone destruction and cytokine expression were determined. Surprisingly, bone destruction was significantly increased in IL-6−/−mice versus that in wild-type mice (by 30%;P< 0.001). In a second experiment, the effects of chronic (IL-6−/−) IL-6 deficiency and short-term IL-6 deficiency induced by in vivo antibody neutralization were determined. Both IL-6−/−(30%;P< 0.001) and anti-IL-6 antibody-treated mice (40%;P< 0.05) exhibited increased periapical bone resorption, compared to wild-type controls. The increased bone resorption in IL-6-deficient animals correlated with increases in osteoclast numbers, as well as with elevated expression of bone-resorptive cytokines IL-1α and IL-1β, in periapical lesions and with decreased expression of the anti-inflammatory cytokine IL-10. These data demonstrate that endogenous IL-6 expression has significant anti-inflammatory effects in modulating infection-stimulated bone destruction in vivo.
Combinatorial Library Cloning of Human Antibodies toStreptococcus pneumoniaeCapsular Polysaccharides: Variable Region Primary Structures and Evidence for Somatic Mutation of Fab Fragments Specific for Capsular Serotypes 6B, 14, and 23F
Authors: Lucas Alexander H. , Moulton Karen D. , Tang Vanessa R. , Reason Donald C.
Published: July 2002

Antibodies specific for capsular polysaccharides play a central role in immunity to encapsulatedStreptococcus pneumoniae, but little is known about their genetics or the variable (V) region polymorphisms that affect their protective function. To begin to address these issues, we used combinatorial library cloning to isolate pneumococcal polysaccharide (PPS)-specific Fab fragments from two vaccinated adults. We determined complete V region primary structures and performed antigen binding analyses of seven Fab fragments specific for PPS serotype 6B, 14, or 23F. Fabs were of the immunoglobulin G2 or A isotype. Several VHIII gene segments (HV 3-7, 3-15, 3-23, and 3-11) were identified. VLregions were encoded by several κ genes (KV 4-1, 3-15, 2-24, and 2D-29) and a λ gene (LV 1-51). Deviation of the VHand VLregions from their assigned germ line counterparts indicated that they were somatically mutated. Fabs of the same serotype specificity isolated from a single individual differed in affinity, and these differences could be accounted for either by the extent of mutation among clonal relatives or by usage of different V-region genes. Thus, functionally disparate anti-PPS antibodies can arise within individuals both by activation of independent clones and by intraclonal somatic mutation. For one pair of clonally related Fabs, the more extensively mutated VHwas associated with lower affinity for PPS 14, a result suggesting that somatic mutation could lead to diminished protective efficacy. These findings indicate that the PPS repertoire in the adult derives from memory B-cell populations that have class switched and undergone extensive hypermutation.
Non-Major Histocompatibility Complex Control of Antibody Isotype and Th1 versus Th2 Cytokines during Experimental Infection of Mice withMycobacterium avium
Authors: Nagabhushanam Vijaya , Cheers Christina
Published: July 2002

Infection of different strains of mice withMycobacterium aviumhas revealed genetic control of the immunoglobulin isotype induced and of the balance between Th1 and Th2 cytokines. Female BALB/c or C57BL/10 mice were infected intranasally with 105M. aviumorganisms. The antibody response was measured over 18 weeks by enzyme-linked immunosorbent assay and Western blotting, while numbers of cytokine-producing cells were assessed at 12 to 15 weeks by ELISPOT assay. Upon infection, C57BL/10 mice produced a clear Th1 response with strong gamma interferon (IFN-γ) production, no interleukin-4 (IL-4), and almost entirely immunoglobulin G2a (IgG2a) antibody. In contrast, BALB/c mice developed T cells producing IL-4, as well as those producing IFN-γ, while the antibody response was a mixture of IgG1 and IgG2a. Antibodies from BALB/c mice were also able to recognize a greater range of antigens than were C56BL/10 mice. B10D2 mice, which carry the BALB/c major histocompatibility complex haplotype on a C57BL/10 background, followed the C57BL/10 cytokine pattern. Mice infected withListeria monocytogenesdid not show a similar response dichotomy.
Preexposure of Murine Macrophages to CpG Oligonucleotide Results in a Biphasic Tumor Necrosis Factor Alpha Response to Subsequent Lipopolysaccharide Challenge
Authors: Crabtree Traves D. , Jin Long , Raymond Daniel P. , Pelletier Shawn J. , Houlgrave C. Webster , Gleason Thomas G. , Pruett Timothy L. , Sawyer Robert G.
Published: July 2002

Bacterial DNA and synthetic oligonucleotides containing CpG sequences (CpG-DNA and CpG-ODN) provoke a proinflammatory cytokine response (tumor necrosis factor alpha [TNF-α], interleukin-12 [IL-12], and IL-6) and increased mortality in lipopolysaccharide (LPS)-challenged mice via a TNF-α-mediated mechanism. It was hypothesized that preexposure of macrophages to CpG-ODN would result in an increased TNF-α response to subsequent LPS challenge in vitro. Using the murine macrophage cell line RAW 264.7, we demonstrated both a rapid proinflammatory cytokine response (TNF-α) and a delayed inhibitory cytokine response (IL-10) with CpG-ODN. Preexposure of macrophages to CpG-ODN for brief periods (1 to 3 h) augmented TNF-α secretion and mRNA accumulation following subsequent LPS challenge (1 μg/ml). However, prolonged preexposure to CpG-ODN (6 to 9 h) resulted in suppression of the TNF-α protein and mRNA response to LPS. The addition of anti-IL-10 antibody to CpG-ODN during preexposure resulted in an increase in the LPS-induced TNF-α response over that induced by CpG-ODN preexposure alone. Thus, while brief preexposure of macrophages to CpG-ODN augments the proinflammatory cytokine response to subsequent LPS challenge, prolonged preexposure elicits IL-10 production, which inhibits the TNF-α response. Although the initial proinflammatory effects of CpG-DNA are well established, the immune response to CpG-DNA may also include autocrine or paracrine feedback mechanisms, leading to a complex interaction of proinflammatory and inhibitory cytokines.
DNA from Protozoan ParasitesBabesia bovis, Trypanosoma cruzi, andT. bruceiIs Mitogenic for B Lymphocytes and Stimulates Macrophage Expression of Interleukin-12, Tumor Necrosis Factor Alpha, and Nitric Oxide
Authors: Shoda Lisl K. M. , Kegerreis Kimberly A. , Suarez Carlos E. , Roditi Isabel , Corral Ricardo S. , Bertot Gustavo M. , Norimine Junzo , Brown Wendy C.
Published: July 2002

The activation of innate immune responses by genomic DNA from bacteria and several nonvertebrate organisms represents a novel mechanism of pathogen recognition. We recently demonstrated the CpG-dependent mitogenic activity of DNA from the protozoan parasiteBabesia bovisfor bovine B lymphocytes (W. C. Brown, D. M. Estes, S. E. Chantler, K. A. Kegerreis, and C. E. Suarez, Infect. Immun. 66:5423–5432, 1998). However, activation of macrophages by DNA from protozoan parasites has not been demonstrated. The present study was therefore conducted to determine whether DNA from the protozan parasitesB. bovis, Trypanosoma cruzi, andT. bruceiactivates macrophages to secrete inflammatory mediators associated with protective immunity. DNA fromEscherichia coliand all three parasites stimulated B-lymphocyte proliferation and increased macrophage production of interleukin-12 (IL-12), tumor necrosis factor alpha (TNF-α), and nitric oxide (NO). Regulation of IL-12 and NO production occurred at the level of transcription. The amounts of IL-12, TNF-α, and NO induced byE. coliand protozoal DNA were strongly correlated (r2> 0.9) with the frequency of CG dinucleotides in the genome, and immunostimulation by DNA occurred in the orderE. coli≥T. cruzi>T. brucei>B. bovis. Induction of inflammatory mediators byE. coli, T. brucei, andB. bovisDNA was dependent on the presence of unmethylated CpG dinucleotides. However, at high concentrations,E. coliandT. cruziDNA-mediated macrophage activation was not inhibited following methylation. The recognition of protozoal DNA by B lymphocytes and macrophages may provide an important innate defense mechanism to control parasite replication and promote persistent infection.
Proinflammatory and Proapoptotic Activities Associated withBordetella pertussisFilamentous Hemagglutinin
Authors: Abramson Tzvia , Kedem Hassya , Relman David A.
Published: July 2002

Filamentous hemagglutinin (FHA) is a dominant cell surface-associatedBordetella pertussisadhesin. Recognition that this protein is secreted in significant amounts and that bacterial adhesins may have other actvities, prompted an assessment of FHA effects on human macrophages. Incubation of human macrophage-like U937 cells with preparations of FHA resulted in dose-dependent cytotoxicity, with death of 95% of treated cells after 24 h. Based on the use of four independent methods, death of these cells could be largely attributed to apoptosis. FHA-associated apoptosis was also observed in THP-1 macrophage-like cells, fresh human peripheral blood monocyte-derived macrophages (MDM), and BEAS-2B human bronchial epithelial cells. Infection of MDM with wild-typeB. pertussisresulted in apoptosis within 6 h, while infection with an FHA-deficient derivative strain was only 50% as effective. FHA-associated cytotoxicity was preceded by host cell secretion of tumor necrosis factor alpha (TNF-α), a potential proapoptotic factor. However, pretreatment of cells with a neutralizing anti-TNF-α monoclonal antibody inhibited only 16% of the FHA-associated apoptosis. On the other hand, a blocking monoclonal antibody directed against TNF-α receptor 1 inhibited FHA-associated apoptosis by 47.7% (P= 0.0001), suggesting that this receptor may play a role in the death pathway activated by FHA. Our in vitro data indicate that secreted and cell-associated FHA elicits proinflammatory and proapoptotic responses in human monocyte-like cells, MDM, and bronchial epithelial cells and suggest a previously unrecognized role for this prominent virulence factor in theB. pertussis-host interaction.
Vacuolating Cytotoxin of
Helicobacter pylori
Induces Apoptosis in the Human Gastric Epithelial Cell Line AGS
Authors: Kuck Dirk , Kolmerer Bernhard , Iking-Konert Christof , Krammer Peter H. , Stremmel Wolfgang , Rudi Jochen
Published: July 2002

Helicobacter pylori
induces cell death by apoptosis. However, the apoptosis-inducing factor is still unknown. The virulence factor vacuolating cytotoxin A (VacA) is a potential candidate, and thus its role in apoptosis induction was investigated in the human gastric epithelial cell line AGS. The supernatant from the
vacA
wild-type strain P12 was able to induce apoptotic cell death, whereas the supernatant from its isogenic mutant strain P14 could not. That VacA was indeed the apoptosis-inducing factor was demonstrated further by substantial reduction of apoptosis upon treatment of AGS cells with a supernatant specifically depleted of native VacA. Furthermore, a recombinant VacA produced in
Escherichia coli
was also able to induce apoptosis in AGS cells but failed to induce cellular vacuolation. These findings demonstrate that the vacuolating cytototoxin of
H. pylori
is a bacterial factor capable of inducing apoptosis in gastric epithelial cells.
Deficient Humoral Responses Underlie Susceptibility toToxoplasma gondiiin CD4-Deficient Mice
Authors: Johnson Lawrence L. , Sayles Peter C.
Published: July 2002

Resistance to infection withToxoplasma gondiiwas studied in mice lacking CD4 expression. Such mice developed more brain cysts and survived for a shorter time than did wild-type controls after peroral infection with ME49 cysts. After immunization with the ts-4 strain ofT. gondii, CD4-deficient mice exhibited impaired resistance to a challenge infection with virulent RH tachyzoites. Thus, deficient CD4 expression increases the susceptibility of mice to a primary peroralT. gondiiinfection with cysts and impairs their ability to be successfully vaccinated. CD8+T cells from blood or spleens ofToxoplasma-infected, CD4-deficient mice expressed markers of activation at frequencies similar to those of infected wild-type mice. Production of IFN-γ in vitro was moderately depressed, and levels ofToxoplasma-specific immunoglobulin G2a in serum were substantially lower than in wild-type mice. Administration ofToxoplasma-immune serum to ts-4-vaccinated CD4-deficient mice significantly improved their resistance to RH challenge. Also, the survival of CD4-deficient mice chronically infected with ME49 was significantly prolonged by administration of immune serum. These results demonstrate that in addition to CD8+T cells and IFN-γ, which are known to be critical for resistance, CD4+cells also contribute significantly to protection against chronicT. gondiiinfections and against challenge infections with highly virulent tachyzoites in immunized mice via their role as helper cells for production of isotype-switched antibodies.
Cytotoxic Activities of
Leptospira interrogans
Hemolysin SphH as a Pore-Forming Protein on Mammalian Cells
Authors: Lee Seoung Hoon , Kim Sangduk , Park Seung Chul , Kim Min Ja
Published: July 2002

Leptospirosis is a spirochetal zoonosis that causes an acute febrile systemic illness in humans.
Leptospira
sp. hemolysins have been shown to be virulence factors for the pathogenesis of leptospirosis. Previously, we cloned a hemolysin SphH of
Leptospira interrogans
serovar lai, a homologue of
L. borgpetersenii
sphingomyelinase (SphA), from a genomic library (S. H. Lee, K. A. Kim, Y. K. Kim, I. W. Seong, M. J. Kim, and Y. J. Lee, Gene 254:19–28, 2000).
Escherichia coli
lysate harboring the
sphH
showed high hemolytic activities on sheep erythrocytes. However, it neither showed sphingomyelinase nor phospholipase activities, in contrast to SphA which was known to have sphingomyelinase activity. Interestingly, the SphH-mediated hemolysis on erythrocytes was osmotically protected by PEG 5000, suggesting that the SphH might have caused pore formation on the erythrocyte membrane. In the present study, we have prepared the
Leptospira
hemolysin SphH and investigated its hemolytic and cytotoxic activities on mammalian cells. SphH was shown to be a pore-forming protein on several mammalian cells: When treated with the SphH, the sheep erythrocyte membranes formed pores, which were morphologically confirmed by transmission electron microscopy. Furthermore, the SphH-mediated cytotoxicities on mammalian cells were demonstrated by the release of LDH and by inverted microscopic examinations. Finally, the immune serum against the full-length hemolysin could effectively neutralize the SphH-mediated hemolytic and cytotoxic activities. In conclusion, these results suggest that the virulence of
Leptospira
SphH was due to the pore formation on mammalian cell membranes.
Increased Activation and Oligoclonality of Peripheral CD8+T Cells in the Chronic Human Helminth Infection Alveolar Echinococcosis
Authors: Manfras Burkhard J. , Reuter Stefan , Wendland Thomas , Kern Peter
Published: July 2002

Alveolar echinococcosis (AE) in humans is a chronic disease characterized by slowly expanding liver lesions. Cellular immunity restricts the spreading of the extracellular pathogen, but functional contributions of CD4+and CD8+T cells are not defined. Here we studied ex vivo the phenotype and function of circulating T-cell subsets in AE patients by means of flow cytometry, T-cell receptor spectratyping, and lymphocyte proliferation. AE patients with parasitic lesions displayed a significant increase of activation of predominantly CD8+T cells compared to healthy controls and AE patients without lesions. In vitro, proliferative T-cell responses to polyclonal stimulation with recall antigens andEchinococcus multilocularisvesicular fluid antigen were sustained during chronic persisting infection in all AE patients. Only in AE patients with parasitic lesions did T-cell receptor spectratyping reveal increased oligoclonality of CD8+but not CD4+T cells, suggesting a persistent antigenic drive for CD8+T cells with subsequent proliferation of selected clonotypes. Thus, our data provide strong evidence for an active role of CD8+T cells in AE.
Bacteriophage φYeO3-12, Specific for
Yersinia enterocolitica
Serotype O:3, Is Related to Coliphages T3 and T7
Authors: Pajunen Maria , Kiljunen Saija , Skurnik Mikael
Published: July 2002

Bacteriophage φYeO3-12 is a lytic phage of
Yersinia enterocolitica
serotype O:3. The phage receptor is the lipopolysaccharide O chain of this serotype that consists of the rare sugar 6-deoxy-
l
-altropyranose. A one-step growth curve of φYeO3-12 revealed eclipse and latent periods of 15 and 25 min, respectively, with a burst size of about 120 PFU per infected cell. In electron microscopy φYeO3-12 virions showed pentagonal outlines, indicating their icosahedral nature. The phage capsid was shown to be composed of at least 10 structural proteins, of which a protein of 43 kDa was predominant. N-terminal sequences of three structural proteins were determined, two of them showing strong homology to structural proteins of coliphages T3 and T7. The phage genome was found to consist of a double-stranded DNA molecule of 40 kb without cohesive ends. A physical map of the phage DNA was constructed using five restriction enzymes. The phage infection could be effectively neutralized using serum from a rabbit immunized with whole φYeO3-12 particles. The antiserum also neutralized T3 infection, although not as efficiently as that of φYeO3-12. φYeO3-12 was found to share, in addition to the N-terminal sequence homology, several common features with T3, including morphology and nonsubjectibility to F exclusion. The evidence conclusively indicated that φYeO3-12 is the first close relative of phage T3 to be described.
Anaerobic Toluene Catabolism of
Thauera aromatica
: the
bbs
Operon Codes for Enzymes of β Oxidation of the Intermediate Benzylsuccinate
Authors: Leuthner Birgitta , Heider Johann
Published: July 2002

The pathway of anaerobic toluene oxidation to benzoyl coenzyme A (benzoyl-CoA) consists of an initial reaction catalyzed by benzylsuccinate synthase, a glycyl radical enzyme adding the methyl group of toluene to the double bond of a fumarate cosubstrate, and a subsequent β-oxidation pathway of benzylsuccinate. Benzylsuccinate synthase has been studied in some detail, whereas the enzymes participating in β oxidation of benzylsuccinate are unknown. We have investigated these enzymes by analyzing substrate-induced proteins in toluene-grown cells. Toluene-induced proteins were identified and N-terminally sequenced. Nine of these proteins are encoded by an 8.5-kb operon consisting of
bbs
(beta-oxidation of benzylsuccinate) genes whose products are apparently involved in the β-oxidation pathway of benzylsuccinate. Two of the genes,
bbsE
and
bbsF
, code for the subunits of a succinyl-CoA:benzylsuccinate CoA-transferase whose activity was previously detected in toluene-grown
Thauera aromatica
. The
bbsG
gene codes for a specific benzylsuccinyl-CoA dehydrogenase, as confirmed by overexpression of the gene in
Escherichia coli
and detection of enzyme activity. The further enzymes of the pathway are probably encoded by
bbsH
(enoyl-CoA hydratase),
bbsCD
(3-hydroxyacyl-CoA dehydrogenase), and
bbsB
(3-oxoacyl-CoA thiolase). The operon contains two additional genes,
bbsA
and
bbsI
, for which no obvious function could be derived. The
bbs
operon is expressed only in toluene-grown cells and is regulated at the transcriptional level. Promoter mapping revealed a transcription start site upstream of the
bbsA
gene. This represents the first known promoter site in
Thauera
spp.
Production of an Endo-β-N-Acetylglucosaminidase Activity Mediates Growth ofEnterococcus faecalison a High-Mannose-Type Glycoprotein
Authors: Roberts Gretta , Tarelli Edward , Homer Karen A. , Philpott-Howard John , Beighton David
Published: July 2002

Enterococcus faecalisis associated with a high proportion of nosocomial infections; however, little is known of the ability of this organism to proliferate in vivo. The ability of RNase B, a model glycoprotein with a single N-glycosylation site occupied by a family of high-mannose-type glycans (Man5- to Man9-GlcNAc2), to support growth ofE. faecaliswas investigated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of RNase B demonstrated a reduction in the molecular mass of this glycoprotein during bacterial growth. Further analysis by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry revealed that this mass shift was due to the degradation of all high-mannose-type glycoforms to a single N-linkedN-acetylglucosamine residue. High-pH anion-exchange chromatography analysis during exponential growth demonstrated the presence of RNase B-derived glycans in the culture supernatant, indicating the presence of an endoglycosidase activity. The free glycans were eluted with the same retention times as those generated by the action ofStreptomyces plicatusendo-β-N-acetylglucosaminidase H on RNase B. The cleavage specificity was confirmed by MALDI-TOF analysis of the free glycans, which showed glycan species containing only oneN-acetylglucosamine residue. No free glycans were detectable after 5 h of bacterial growth, and we have subsequently demonstrated the presence of mannosidase activity inE. faecalis, which releases free mannose from RNase B-derived glycans. We propose that this deglycosylation of glycoproteins containing high-mannose-type glycans and the subsequent degradation of the released glycans byE. faecalismay play a role in the survival and persistence of this nosocomial pathogen in vivo.
The
gaf
Fimbrial Gene Cluster of
Escherichia coli
Expresses a Full-Size and a Truncated Soluble Adhesin Protein
Authors: Tanskanen Jarna , Saarela Sirkku , Tankka Sanna , Kalkkinen Nisse , Rhen Mikael , Korhonen Timo K. , Westerlund-Wikström Benita
Published: July 2002

The GafD lectin of the G (F17) fimbriae of diarrhea-associated
Escherichia coli
was overexpressed and purified from the periplasm of
E. coli
by affinity chromatography on GlcNAc-agarose. The predicted mature GafD peptide comprises 321 amino acids, but the predominant form of GafD recovered from the periplasm was 19,092 Da in size and corresponded to the 178 N-terminal amino acid residues, as judged by mass spectrometry and amino acid sequencing, and was named ΔGafD. Expression of
gafD
from the cloned
gaf
gene cluster in DegP-, Lon-, and OmpT-deficient recombinant strains did not significantly decrease the formation of ΔGafD. The peptide was also detected in the periplasm of the wild-type
E. coli
strain from which the
gaf
gene cluster originally was cloned. We expressed
gafD
fragments encoding C-terminally truncated peptides. Peptides GafD1-252, GafD1-224, GafD1-189, and the GafD1-178, isolated from the periplasm by affinity chromatography, had apparent sizes closely similar to that of ΔGafD. Only trace amounts of truncated forms with expected molecular sizes were detected in spheroplasts. In contrast, the shorter GafD1-157 peptide was detected in spheroplasts but not in the periplasm, indicating that it was poorly translocated or was degraded by periplasmic proteases. Pulse-chase assays using
gafD
indicated that ΔGafD was processed from GafD and is not a primary translation product. The ΔGafD peptide was soluble by biochemical criteria and exhibited specific binding to GlcNAc-agarose. Inhibition assays with mono- and oligosaccharides gave a similar inhibition pattern in the hemagglutination by the G-fimbria-expressing recombinant
E. coli
strain and in the binding of [
14
C]ΔGafD to GlcNAc-agarose. ΔGafD bound specifically to laminin, a previously described tissue target for the G fimbria. Our results show that a soluble, protease-resistant subdomain of GafD exhibits receptor-binding specificity similar to that for intact G fimbriae and that it is formed when
gafD
is expressed alone or from the
gaf
gene cluster.
Analyses of
mrp
Genes during
Myxococcus xanthus
Development
Authors: Sun Hong , Shi Wenyuan
Published: July 2002

Myxococcus xanthus
is a gram-negative soil bacterium that undergoes development under starvation conditions. Our previous study identified a new genetic locus,
mrp
, which is required for both fruiting body formation and sporulation. The locus encodes two transcripts:
mrpAB
, which consists of a histidine kinase and an NtrC-like response regulator, and
mrpC
, a cyclic AMP receptor protein family transcription activator. In this study, we used genetic and biochemical analyses to investigate the possible interactions between the
mrp
genes and other known developmental genes and events. These studies show that the
mrp
genes possibly function after A-signaling and (p)ppGpp but before C-signaling and that they regulate various early and late developmental genes and events.
Postsegregational Killing Mediated by the P1 Phage “Addiction Module”
phd-doc
Requires the
Escherichia coli
Programmed Cell Death System
mazEF
Authors: Hazan Ronen , Sat Boaz , Reches Myriam , Engelberg-Kulka Hanna
Published: July 2002

“Addiction modules” consist of two genes; the product of the second is long lived and toxic, while the product of the first is short lived and antagonizes the lethal action of the toxin. The extrachromosomal addiction module
phd-doc
, located on the P1 prophage, is responsible for the postsegregational killing effect (death of plasmid-free cells). The
Escherichia coli
chromosomal addiction module analogue,
mazEF
, is responsible for the induction of programmed cell death. Here we show that the postsegregational killing mediated by the P1
phd-doc
module depends on the presence of the
E. coli mazEF
system. In addition, we demonstrate that under conditions of postsegregational killing, mediated by
phd-doc
, protein synthesis of
E. coli
is inhibited. Based on our findings, we suggest the existence of a coupling between the
phd-doc
and
mazEF
systems.
The DevT Protein Stimulates Synthesis of FruA, a Signal Transduction Protein Required for Fruiting Body Morphogenesis in
Myxococcus xanthus
Authors: Boysen Anders , Ellehauge Eva , Julien Bryan , Søgaard-Andersen Lotte
Published: July 2002

Fruiting body formation in
Myxococcus xanthus
involves three morphologic stages—rippling, aggregation, and sporulation—all of which are induced by the cell surface-associated C-signal. We analyzed the function of the DevT protein, a novel component in the C-signal response pathway. A mutant carrying an in-frame deletion in the
devT
gene displays delayed aggregation and a cell autonomous sporulation defect, whereas it remains rippling proficient. To further define the function of DevT, the methylation pattern of FrzCD, a cytoplasmic methyl-accepting chemotaxis protein homologue, was examined in the Δ
devT
mutant, and we found that DevT is required for methylation of FrzCD during development. Specifically, DevT was found to be required for the C-signal-dependent methylation of FrzCD. The Δ
devT
mutant produced wild-type levels of C-signal. However, accumulation of the FruA response regulator protein, which is essential for the execution of the C-signal-dependent responses, was reduced in the Δ
devT
mutant. The DevT protein was found to stimulate the developmentally activated transcription of the
fruA
gene. Epistasis analyses indicate that DevT acts independently of the A- and E-signals to stimulate
fruA
transcription. These findings suggest that the developmental defects of the Δ
devT
mutant are associated with a lack of FruA to ensure a proper response to the C-signal during the aggregation and sporulation stages of development.
Enzyme-Linked Immunosorbent Assays for Detection of Antibodies to Ebola and Marburg Viruses Using Recombinant Nucleoproteins
Authors: Saijo Masayuki , Niikura Masahiro , Morikawa Shigeru , Ksiazek Thomas G. , Meyer Richard F. , Peters Clarence J. , Kurane Ichiro
Published: July 2002

The full-length nucleoprotein (NP) of Ebola virus (EBO) was expressed as a His-tagged recombinant protein (His-EBO-NP) by a baculovirus system. Carboxy-terminal halves of NPs of EBO and Marburg virus (MBG) were expressed as glutathione
S
-transferase-tagged recombinant proteins in an
Escherichia coli
system. The antigenic regions on the NPs of EBO and MBG were determined by both Western blotting and enzyme-linked immunosorbent assay (ELISA) to be located on the C-terminal halves. The C-terminal 110 and 102 amino acids of the NPs of EBO and MBG, respectively, possess strong antigenicity. The full-length NP of EBO was strongly expressed in insect cells upon infection with the recombinant baculovirus, while expression of the full-length NP of MBG was weak. We developed an immunoglobulin G (IgG) ELISA using His-EBO-NP and the C-terminal halves of the NPs of EBO and MBG as antigens. We evaluated the IgG ELISA for the ability to detect IgG antibodies to EBO and MBG, using human sera collected from EBO and MBG patients. The IgG ELISA with the recombinant NPs showed high sensitivity and specificity in detecting EBO and MBG antibodies. The results indicate that ELISA systems prepared with the recombinant NPs of EBO and MBG are valuable tools for the diagnosis of EBO and MBG infections and for seroepidemiological field studies.
Identification of New Repetitive Element in
Leptospira interrogans
Serovar Copenhageni and Its Application to PCR-Based Differentiation of
Leptospira
Serogroups
Authors: Barocchi Michele A. , Ko Albert I. , Ferrer Suzana Ramos , Faria Marcos Tucunduva , Reis Mitermayer Galvão , Riley Lee W.
Published: July 2002

A new repetitive DNA element was identified in an isolate of
Leptospira interrogans
serovar copenhageni from a patient in Salvador, Brazil. A
Sau
3A genomic library from this strain was constructed and screened for repetitive DNA elements. An insert of 438 bp (Rep1) from one library clone hybridized to multiple chromosomal DNA fragments resolved electrophoretically after digestion with
Bam
HI,
Hin
dIII, and
Mfe
I. A single oligonucleotide primer, designated iRepl, was designed to generate multiple PCR amplicons of various electrophoretic mobilities in a PCR typing method. The method distinguished strains belonging to the eight pathogenic and three saprophytic species of the genus
Leptospira
. Clinical isolates obtained during urban epidemics between 1996 and 1998 in Salvador, Brazil, were analyzed by this PCR method. Although the iRep1 primer was unable to discriminate strains among
L. interrogans
serovar copenhageni isolates, it was able to differentiate strains belonging to different species and serogroups of
Leptospira
identified in Salvador. This PCR-based method may provide a faster and less expensive alternative to serologic tests used in reference laboratories.
Prospective Multicenter Clinical Evaluation of AMPLICOR and COBAS AMPLICOR Hepatitis C Virus Tests
Authors: Nolte Frederick S. , Fried Michael W. , Shiffman Mitchell L. , Ferreira-Gonzalez Andrea , Garrett Carlton T. , Schiff Eugene R. , Polyak Stephen J. , Gretch David R.
Published: July 2002

We conducted a multicenter clinical evaluation of the second versions of the manual AMPLICOR and the semiautomated COBAS AMPLICOR tests for hepatitis C virus (HCV) RNA (Roche Molecular Systems, Inc., Pleasanton, Calif.). The performance characteristics of these HCV RNA tests for diagnosis of active viral infection were determined by comparison to anti-HCV serological test results, alanine aminotransferase levels, and liver biopsy histology results. A total of 878 patients with clinical or biochemical evidence of liver disease were enrolled at four hepatology clinics. A total of 1,089 specimens (901 serum and 188 plasma) were tested with the AMPLICOR test. Sensitivity compared to serology was 93.1% for serum and 90.6% for plasma. The specificity was 97% for serum and 93.1% for plasma. A total of 1,084 specimens (896 serum and 188 plasma) were tested with the COBAS test. Sensitivities for serum and plasma were the same as with the AMPLICOR test. The specificity was 97.8% for serum and 96.6% for plasma. Of the 69 specimens with false-positive and false-negative AMPLICOR test results relative to those of serology, alternative primer set (APS) reverse transcription (RT)-PCR analysis showed that the AMPLICOR test provided the correct result relative to the specimens containing HCV RNA in 64 (92.7%) specimens. Similarly, 66 of 67 (98.5%) false-positive and false-negative COBAS test results were determined to be correct by APS RT-PCR analysis. There were no substantive differences in clinical performances between study sites, patient groups, specimen types, storage conditions (−20 to −80°C versus 2 to 8°C), or anticoagulants (EDTA versus acid citrate dextrose) for either test. Both tests showed >99% reproducibility within runs, within sites, and overall. We conclude that these tests can reliably detect the presence of HCV RNA, as evidence of active infection, in patients with clinical or biochemical evidence of liver disease.
Rapid Identification of Mycobacteria to the Species Level Using INNO-LiPA Mycobacteria, a Reverse Hybridization Assay
Authors: Suffys P. N. , da Silva Rocha A. , de Oliveira M. , Dias Campos C. E. , Werneck Barreto A. M. , Portaels F. , Rigouts L. , Wouters G. , Jannes G. , van Reybroeck G. , Mijs W. , Vanderborght B.
Published: July 2002

INNO-LiPA Mycobacteria (LiPA; Innogenetics, Zwijnaarde, Belgium) is a kit for the simultaneous detection and identification of
Mycobacterium
species in culture and identifies the
Mycobacterium tuberculosis
complex, the
M. avium
complex (MAC), and the following
Mycobacterium
species:
M. kansasii
,
M. avium
,
M. intracellulare
,
M. scrofulaceum
,
M. gordonae
,
M. xenopi
, and the
M. chelonae-M. abscessus
complex. The assay, which targets the 16S-23S rRNA spacer region, was evaluated on 157 mycobacterial strains that had been identified by conventional techniques and PCR-restriction enzyme analysis of the
hsp65
gene (PRA). Forty-seven reference strains consisting of 37 different species and 110 human clinical isolates were submitted to the test, and all were hybridized with the
Mycobacterium
genus probe (MYC) on the LiPA strip (100% sensitivity). Ninety-four isolates hybridized to their corresponding species- or complex-specific probes; only one isolate phenotypically identified as
M. gordonae
did not react with its specific probe (99.4% accuracy). Thirty-seven MAC strains were phenotypically identified to the complex level and to the species level by LiPA as
M. avium
(
n
= 18) or
M. intracellulare
(
n
= 7) or as belonging to the
M. avium
-
M. intracellulare
-
M. scrofulaceum
complex (
n
= 12). Of the last 12 strains, 10 had
M. avium
PRA patterns and 2 had
M. intracellulare
PRA patterns. Three isolates that had been identified as a single species by conventional identification were proven to be mixed cultures by the LiPA assay. The whole procedure can be performed in 1 working day, starting with the supernatant of a small amount of bacterial mass that had been treated by freezing and then boiling.
Identification of B-Cell Epitope of Dengue Virus Type 1 and Its Application in Diagnosis of Patients
Authors: Wu Han-Chung , Huang Yue-Ling , Chao Ting-Ting , Jan Jia-Tsrong , Huang Jau-Ling , Chiang Hsien-Yuan , King Chwan-Chuen , Shaio Men-Fang
Published: July 2002

Using a serotype-specific monoclonal antibody (MAb) of dengue virus type 1 (DEN-1), 15F3-1, we identified the B-cell epitope of DEN-1 from a random peptide library displayed on phage. Fourteen immunopositive phage clones that bound specifically to MAb 15F3-1 were selected. These phage-borne peptides had a consensus motif of HxYaWb (a = S/T, b = K/H/R) that mimicked the sequence HKYSWK, which corresponded to amino acid residues 111 to 116 of the nonstructural protein 1 (NS1) of DEN-1. Among the four synthetic peptides corresponding to amino acid residues 110 to 117 of the NS1 of DEN-1, -2, -3, and -4, only one peptide, EHKYSWKS (P14M) of DEN-1, was found to bind to 15F3-1 specifically. Furthermore, P14M was shown to inhibit the binding of phage particles to 15F3-1 in a competitive inhibition assay. Histidine111(His111) was crucial to the binding of P14M to 15F3-1, since its binding activity dramatically reduced when it changed to leucine111(Leu111). This epitope-based peptide demonstrated its clinical diagnostic potential when it reacted with a high degree of specificity with serum samples obtained from both DEN-1-infected rabbits and patients. Based on these observations, our DEN-1 epitope-based serologic test could be useful in laboratory viral diagnosis and in understanding the pathogenesis of DEN-1.
Evaluation of Clarithromycin Resistance and
cagA
and
vacA
Genotyping of
Helicobacter pylori
Strains from the West of Ireland Using Line Probe Assays
Authors: Ryan Kieran A. , van Doorn Leen-Jan , Moran Anthony P. , Glennon Maura , Smith Terry , Maher Majella
Published: July 2002

The prevalence of clarithromycin resistance-associated mutations, the cytotoxin-associated gene (
cagA
), and the various vacuolating cytotoxin (
vacA
) genotypes was determined in 50 gastric biopsy specimens from
Helicobacter pylori
-infected patients, using line probe assays. The clarithromycin resistance-associated mutation A2143G was detected in
H. pylori
strains from 26% of the specimens, which suggested that the high rate of
H. pylori
treatment failure in Ireland may be partly attributable to the presence of these mutations. All strains examined carried the
vacA
s1 genotype, and 76% were
cagA
positive. Of these 50 specimens, 13 (26%) carried
H. pylori
strains with
vacA
midregion genotype m1, 29 (58%) carried strains that were m2, 1 (2%) was infected by a strain that was positive for both m1 and m2, and 7 (14%) carried strains that could not be typed.
Evidence of In Vivo Transfer of a Plasmid Encoding the Extended-Spectrum β-Lactamase TEM-24 and Other Resistance Factors among Different Members of the Family
Enterobacteriaceae
Authors: Neuwirth Catherine , Siebor Eliane , Pechinot Andre , Duez Jean-Marie , Pruneaux Michele , Garel Frederic , Kazmierczak Antoine , Labia Roger
Published: July 2002

The epidemiological study of several multidrug-resistant
Enterobacteriaceae
isolated from five patients demonstrated in vivo dissemination of a 100-kb plasmid encoding the extended-spectrum β-lactamase TEM-24 from a clonal strain of
Enterobacter aerogenes
to different strains of
Klebsiella pneumoniae, Escherichia coli, Proteus vulgaris, Proteus mirabilis
, and
Serratia marcescens
.
Detection of Herpes Simplex Virus DNA in Genital and Dermal Specimens by LightCycler PCR after Extraction using the IsoQuick, MagNA Pure, and BioRobot 9604 Methods
Authors: Espy Mark J. , Rys Paul N. , Wold Arlo D. , Uhl James R. , Sloan Lynne M. , Jenkins Greg D. , Ilstrup Duane M. , Cockerill Frank R. , Patel Robin , Rosenblatt Jon E. , Smith Thomas F.
Published: July 2002

We evaluated two automated systems, MagNA Pure (Roche Molecular Biochemicals, Indianapolis, Ind.) and BioRobot 9604 (Qiagen, Inc., Chatsworth, Calif.) as effective replacements for the manual IsoQuick method (Orca Research, Inc., Bothell, Wash.) for extraction of herpes simplex virus (HSV) DNA from dermal and genital tract specimens prior to analysis by LightCycler PCR. Of 198 specimens (152 genital, 46 dermal), 92 (46.2%) were positive for HSV DNA by LightCycler PCR after automated extraction of specimens with either the MagNA Pure or BioRobot 9604 instrument. The manual IsoQuick method yielded HSV DNA (total
n
= 95) from three additional specimens that were negative by the automated method (
P
= 0.25, sign test). Although the mean numbers of LightCycler PCR cycles required to reach positivity differed statistically significantly among all three of the methods of extraction, the estimated means differed by no more than 1.5 cycles (
P
< 0.05). Seventy (76%) of the 92 specimens that were LightCycler PCR positive by all three extraction methods were also positive by shell vial cell culture assay. HSV DNA was detected by a lower LightCycler PCR cycle number (26.1 cycles) in specimens culture positive for the virus than in culture-negative samples (33.3 cycles) (
P
< 0.0001). The manual IsoQuick and automated MagNA Pure and BioRobot 9604 methods provide standardized, reproducible extraction of HSV DNA for LightCycler PCR. The decision to implement a manual versus an automated procedure depends on factors such as costs related to the number of specimens processed rather than on the minimal differences in the technical efficiency of extraction of nucleic acids among these methods.
Use of Pulsed-Field Gel Electrophoresis, Enterobacterial Repetitive Intergenic Consensus Typing, and Automated Ribotyping To Assess Genomic Variability among Strains of Nontypeable
Haemophilus influenzae
Authors: Pettigrew M. M. , Foxman B. , Ecevit Z. , Marrs C. F. , Gilsdorf J.
Published: July 2002

We compared 75 nontypeable (NT)
Haemophilus influenzae
isolates by pulsed-field gel electrophoresis (PFGE), enterobacterial repetitive intergenic consensus (ERIC)-PCR, and automated ribotyping. PFGE was the most discriminatory of the techniques. ERIC-PCR provides a useful screen but should not replace other techniques as the sole method to group NT
H. influenzae
strains.
Biochemical and Susceptibility Tests Useful for Identification of Nonfermenting Gram-Negative Rods
Authors: Laffineur Kim , Janssens Michèle , Charlier Jacqueline , Avesani Véronique , Wauters Georges , Delmée Michel
Published: July 2002

Six hundred nineteen strains of nonfermenting gram-negative rods were tested for alkaline phosphatase, benzyl-arginine arylamidase, pyrrolidonyl arylamidase, ethylene glycol acidification, and susceptibility to desferrioxamine and colistin. The results were highly discriminant. Therefore, the proposed tests may be helpful for the identification of this group of organisms.
Natural Genetic Exchanges between Vaccine and Wild Poliovirus Strains in Humans
Authors: Guillot Sophie , Caro Valérie , Cuervo Nancy , Korotkova Ekaterina , Combiescu Mariana , Persu Ana , Aubert-Combiescu Andrei , Delpeyroux Francis , Crainic Radu
Published: July 2002

In a previous study of poliovirus vaccine-derived strains isolated from patients with vaccine-associated paralytic poliomyelitis (VAPP) (9, 11), we reported that a high proportion (over 50%) of viruses had a recombinant genome. Most were intertypic vaccine/vaccine recombinants. However, some had restriction fragment length polymorphism (RFLP) profiles different from those of poliovirus vaccine strains. We demonstrate here that five such recombinants, of 88 VAPP strains examined, carried sequences of wild (nonvaccine) origin. To identify the parental wild donor of these sequences, we used RFLP profiles and nucleotide sequencing to look for similarity in the 3D polymerase-coding region of 61 wild, cocirculating poliovirus isolates (43 type 1, 16 type 2, and 2 type 3 isolates). In only one case was the donor identified, and it was a wild type 1 poliovirus. For the other four vaccine/wild recombinants, the wild parent could not be identified. The possibility that the wild sequences were of a non-poliovirus-enterovirus origin could not be excluded. Another vaccine/wild recombinant, isolated in Belarus from a VAPP case, indicated that the poliovirus vaccine/wild recombination is not an isolated phenomenon. We also found wild polioviruses (2 of 15) carrying vaccine-derived sequences in the 3′ moiety of their genome. All these results suggest that genetic exchanges with wild poliovirus and perhaps with nonpoliovirus enteroviruses, are also a natural means of evolution for poliovirus vaccine strains.
Passage of Classical Swine Fever Virus in Cultured Swine Kidney Cells Selects Virus Variants That Bind to Heparan Sulfate due to a Single Amino Acid Change in Envelope Protein E
rns
Authors: Hulst M. M. , van Gennip H. G. P. , Moormann R. J. M.
Published: July 2002

Infection of cells with Classical swine fever virus (CSFV) is mediated by the interaction of envelope glycoprotein E
rns
and E2 with the cell surface. In this report we studied the role of the cell surface glycoaminoglycans (GAGs), chondroitin sulfates A, B, and C (CS-A, -B, and -C), and heparan sulfate (HS) in the initial binding of CSFV strain Brescia to cells. Removal of HS from the surface of swine kidney cells (SK6) by heparinase I treatment almost completely abolished infection of these cells with virus that was extensively passaged in swine kidney cells before it was cloned (clone C1.1.1). Infection with C1.1.1 was inhibited completely by heparin (a GAG chemically related to HS but sulfated to a higher extent) and by dextran sulfate (an artificial highly sulfated polysaccharide), whereas HS and CS-A, -B, and -C were unable to inhibit infection. Bound C1.1.1 virus particles were released from the cell surface by treatment with heparin. Furthermore, C1.1.1 virus particles and CSFV E
rns
purified from insect cells bound to immobilized heparin, whereas purified CSFV E2 did not. These results indicate that initial binding of this virus clone is accomplished by the interaction of E
rns
with cell surface HS. In contrast, infection of SK6 cells with virus clones isolated from the blood of an infected pig and minimally passaged in SK6 cells was not affected by heparinase I treatment of cells and the addition of heparin to the medium. However, after one additional round of amplification in SK6 cells, infection with these virus clones was affected by heparinase I treatment and heparin. Sequence analysis of the E
rns
genes of these virus clones before and after amplification in SK6 cells showed that passage in SK6 cells resulted in a change of an Ser residue to an Arg residue in the C terminus of E
rns
(amino acid 476 in the polyprotein of CSFV). Replacement of the E
rns
gene of an infectious DNA copy of C1.1.1 with the E
rns
genes of these virus variants proved that acquisition of this Arg was sufficient to alter an HS-independent virus to a virus that uses HS as an E
rns
receptor.
Influenza Virus Matrix Protein Is the Major Driving Force in Virus Budding
Authors: Gómez-Puertas Paulino , Albo Carmen , Pérez-Pastrana Esperanza , Vivo Amparo , Portela Agustı́n
Published: July 2002

To get insights into the role played by each of the influenza A virus polypeptides in morphogenesis and virus particle assembly, the generation of virus-like particles (VLPs) has been examined in COS-1 cell cultures expressing, from recombinant plasmids, different combinations of the viral structural proteins. The presence of VLPs was examined biochemically, following centrifugation of the supernatants collected from transfected cells through sucrose cushions and immunoblotting, and by electron-microscopic analysis. It is demonstrated that the matrix (M1) protein is the only viral component which is essential for VLP formation and that the viral ribonucleoproteins are not required for virus particle formation. It is also shown that the M1 protein, when expressed alone, assembles into virus-like budding particles, which are released in the culture medium, and that the recombinant M1 protein accumulates intracellularly, forming tubular structures. All these results are discussed with regard to the roles played by the virus polypeptides during virus assembly.