bluuebunny/crossref_metadata_2025_split huggingface dataset:
Modification of Lipid A Biosynthesis in
Neisseria meningitidis lpxL
Mutants: Influence on Lipopolysaccharide Structure, Toxicity, and Adjuvant Activity
Authors: van der Ley Peter , Steeghs Liana , Hamstra Hendrik Jan , ten Hove Jan , Zomer Bert , van Alphen Loek 
Published: July 2002

ABSTRACT

Two genes homologous to
lpxL
and
lpxM
from
Escherichia coli
and other gram-negative bacteria, which are involved in lipid A acyloxyacylation, were identified in
Neisseria meningitidis
strain H44/76 and insertionally inactivated. Analysis by tandem mass spectrometry showed that one of the resulting mutants, termed
lpxL1
, makes lipopolysaccharide (LPS) with penta- instead of hexa-acylated lipid A, in which the secondary lauroyl chain is specifically missing from the nonreducing end of the GlcN disaccharide. Insertional inactivation of the other (
lpxL2
) gene was not possible in wild-type strain H44/76 expressing full-length immunotype L3 lipopolysaccharide (LPS) but could be readily achieved in a
galE
mutant expressing a truncated oligosaccharide chain. Structural analysis of
lpxL2
mutant lipid A showed a major tetra-acylated species lacking both secondary lauroyl chains and a minor penta-acylated species. The
lpxL1
mutant LPS has retained adjuvant activity similar to wild-type meningococcal LPS when used for immunization of mice in combination with LPS-deficient outer membrane complexes from
N. meningitidis
but has reduced toxicity as measured in a tumor necrosis factor alpha induction assay with whole bacteria. In contrast, both adjuvant activity and toxicity of the
lpxL2
mutant LPS are strongly reduced. As the combination of reduced toxicity and retained adjuvant activity has not been reported before for either
lpxL
or
lpxM
mutants from other bacterial species, our results demonstrate that modification of meningococcal lipid A biosynthesis can lead to novel LPS species more suitable for inclusion in human vaccines.
Salmonella
DNA Adenine Methylase Mutants Confer Cross-Protective Immunity
Authors: Heithoff Douglas M. , Enioutina Elena Y. , Daynes Raymond A. , Sinsheimer Robert L. , Low David A. , Mahan Michael J. 
Published: July 2002

ABSTRACT

Salmonella
isolates that lack or overproduce DNA adenine methylase (Dam) elicited a cross-protective immune response to different
Salmonella
serovars. The protection afforded by the
Salmonella enterica
serovar Typhimurium Dam vaccine was greater than that elicited in mice that survived a virulent infection.
S. enterica
serovar Typhimurium Dam mutant strains exhibited enhanced sensitivity to mediators of innate immunity such as antimicrobial peptides, bile salts, and hydrogen peroxide. Also,
S. enterica
serovar Typhimurium Dam
−
vaccines were not immunosuppressive; unlike wild-type vaccines, they failed to induce increased nitric oxide levels and permitted a subsequent robust humoral response to diptheria toxoid antigen in infected mice. Dam mutant strains exhibited a low-grade persistence which, coupled with the nonimmunosuppression and the ectopic protein expression caused by altered levels of Dam, may provide an expanded source of potential antigens in vaccinated hosts.
Diminished Virulence of an Alpha-Toxin Mutant ofStaphylococcus aureusin Experimental Brain Abscesses
Authors: Kielian Tammy , Cheung Ambrose , Hickey William F. 
Published: July 2002

ABSTRACTStaphylococcus aureusis one of the major etiologic agents of brain abscesses in humans, occasionally leading to focal neurological deficits and even death. The objective of the present study was to identify key virulence determinants contributing to the pathogenesis ofS. aureusin the brain using a murine brain abscess model. The importance of virulence factor production in disease development was demonstrated by the inability of heat-inactivatedS. aureusto induce proinflammatory cytokine or chemokine expression or brain abscess formation in vivo. To directly address the contribution of virulence determinants in brain abscess development, the abilities ofS. aureusstrains with mutations in the global regulatory locisarAandagrwere examined. AnS.aureus sarAagrdouble mutant exhibited reduced virulence in vivo, as demonstrated by attenuated proinflammatory cytokine and chemokine expression and bacterial replication. Subsequent studies focused on the expression of factors that are altered in thesarA agrdouble mutant. Evaluation of an alpha-toxin mutant revealed a phenotype similar to that of thesarA agrmutant in vivo, as evidenced by lower bacterial burdens and attenuation of cytokine and chemokine expression in the brain. This suggested that alpha-toxin is a central virulence determinant in brain abscess development. Another virulence mechanism utilized by staphylococci is intracellular survival. Cells recovered from brain abscesses were shown to harborS. aureusintracellularly, providing a means by which the organism may establish chronic infections in the brain. Together, these data identify alpha-toxin as a key virulence determinant for the survival ofS. aureusin the brain.
Host Response to Infection: the Role of CpG DNA in Induction of Cyclooxygenase 2 and Nitric Oxide Synthase 2 in Murine Macrophages
Authors: Ghosh Dipak K. , Misukonis Mary A. , Reich Charles , Pisetsky David S. , Weinberg J. Brice 
Published: July 2002

ABSTRACTDepending on sequence, bacterial and synthetic DNAs can activate the host immune system and influence the host response to infection. The purpose of this study was to determine the abilities of various phosphorothioate oligonucleotides with cytosine-guanosine-containing motifs (CpG DNA) to activate macrophages to produce nitric oxide (NO) and prostaglandin E2(PGE2) and to induce expression of NO synthase 2 (NOS2) and cyclooxygenase 2 (COX2). As little as 0.3 μg of CpG DNA/ml increased NO and PGE2production in a dose- and time-dependent fashion in cells of the mouse macrophage cell line J774. NO and PGE2production was noted by 4 to 8 h after initiation of cultures with the CpG DNA, with the kinetics of NO production induced by CpG DNA being comparable to that induced by a combination of lipopolysaccharide and gamma interferon. CpG DNA-treated J774 cells showed enhanced expression of NOS2 and COX2 proteins as determined by immunoblotting, with the relative potencies of the CpG DNAs generally corresponding to those noted for the induction of NO and PGE2production as well as to those noted for the induction of interleukin-6 (IL-6), IL-12, and tumor necrosis factor. Extracts from CpG DNA-treated cells convertedl-arginine tol-citrulline, but the NOS inhibitorNG-monomethyl-l-arginine (NMMA) inhibited this reaction. The COX2-specific inhibitor NS398 inhibited CpG DNA-induced PGE2production and inhibited NO production to various degrees. The NOS inhibitors NMMA, 1400W, andN-iminoethyl-l-lysine effectively blocked NO production and increased the production of PGE2in a dose-dependent fashion. Thus, analogues of microbial DNA (i.e., CpG DNA) activate mouse macrophage lineage cells for the expression of NOS2 and COX2, with the production of NO and that of PGE2occurring in an interdependent manner.
Serum Resistance in an Invasive, NontypeableHaemophilus influenzaeStrain
Authors: Williams Bryan J. , Morlin Gregory , Valentine Nathan , Smith Arnold L. 
Published: July 2002

ABSTRACTA common feature of many different organisms causing bacteremia is the ability to avoid the bactericidal effects of normal human serum. InHaemophilus influenzaeencapsulated strains are particularly serum resistant; however, we found that a nonencapsulated strain (R2866) isolated from the blood of an immunocompetent child with meningitis who had been successfully immunized withH. influenzaetype b conjugate vaccine was serum resistant. Since serum resistance usually involves circumventing the action of the complement system, we defined the deposition of various complement components on the surfaces of thisH. influenzaestrain (R2866), a nonencapsulated avirulent laboratory strain (Rd), and a virulent type b encapsulated strain (Eagan). Membrane attack complex (MAC) accumulation correlated with the loss of bacterial viability; correspondingly, the rates of MAC deposition on the serum-sensitive strain Rd and the serum-resistant strains differed. Analysis of cell-associated immunoglobulin G (IgG), C1q, C3b, and C5b indicated that serum-resistantH. influenzaeprevents MAC accumulation by delaying the synthesis of C3b through the classical pathway. Among the initiators of the classical pathway, IgG deposition contributes most of the C3 convertase activity necessary to start the cascade ending with MAC deposition. Despite similar IgG binding, strain R2866 delays C3 convertase activity compared to strain Rd. We conclude that strain R2866 can persist in the bloodstream, in part by inhibiting or delaying C3 deposition on the cell surface, escaping complement mediated killing.
Safety and Immunogenicity of Increasing Doses of a
Clostridium difficile
Toxoid Vaccine Administered to Healthy Adults
Authors: Kotloff Karen L. , Wasserman Steven S. , Losonsky Genevieve A. , Thomas William , Nichols Richard , Edelman Robert , Bridwell Margaret , Monath Thomas P. 
Published: July 2002

ABSTRACT

Clostridium difficile
is a major cause of nosocomial diarrhea in industrialized countries. Although most illnesses respond to available therapy, infection can increase morbidity, prolong hospitalization, and produce life-threatening colitis. Vaccines are being explored as an alternative means for protecting high-risk individuals. We assessed the safety, immunogenicity, and dose response of a parenteral vaccine containing
C. difficile
toxoids A and B. Thirty healthy adults were assigned to receive four spaced inoculations on days 1, 8, 30, and 60 with one of three doses of vaccine (6.25, 25, or 100 μg). At each dose level, subjects were randomized, in a double-blind fashion, to receive either the soluble toxoids (
n
= 5) or toxoids adsorbed to alum (
n
= 5). Subjects were monitored for clinical and immunologic responses to vaccination. Vaccination was generally well tolerated, with occasional, usually mild, systemic reactions (abdominal pain, arthralgia, and diarrhea). The most common local reaction, mild arm pain, was reported by all recipients of the toxoid-alum formulation. Nearly all subjects (≥90%) developed vigorous serum antibody responses to both toxins, as measured by immunoglobulin G (IgG) enzyme-linked immunosorbent assay and neutralization of cytotoxicity, whereas fecal IgA increases occurred in approximately 50%. Statistically significant effects of dose and formulation on immunogenicity were not seen, although antibody levels tended to be higher with the alum-adjuvanted formulations and with increasing doses of soluble toxoid. Serum antibody responses among the toxoid-alum group appeared to plateau at 25 μg. We concluded that the
C. difficile
toxoid vaccine is safe and immunogenic in healthy volunteers. Further development as a prophylactic vaccine or for producing
C. difficile
hyperimmune globulin is justified.
Passive Transfer of Immunoglobulin Y Antibody to
Streptococcus mutans
Glucan Binding Protein B Can Confer Protection against Experimental Dental Caries
Authors: Smith Daniel J. , King William F. , Godiska Ronald 
Published: July 2002

ABSTRACT

Active immunization with
Streptococcus mutans
glucan binding protein B (GBP-B) has been shown to induce protection against experimental dental caries. This protection presumably results from continuous secretion of salivary antibody to GBP-B, which inhibits accumulation of
S. mutans
within the oral biofilm. The purpose of this study was to explore the influence of short-term (9- or 24-day) passive oral administration of antibody to
S. mutans
GBP-B on the longer-term accumulation and cariogenicity of
S. mutans
in a rat model of dental caries. Preimmune chicken egg yolk immunoglobulin Y (IgY) or IgY antibody to
S. mutans
GBP-B was supplied in lower (experiment 1) and higher (experiment 2) concentrations in the diet and drinking water of rats for 9 (experiment 1) or 24 (experiment 2) days. During the first 3 days of IgY feeding, all animals were challenged with 5 × 10
6
streptomycin-resistant
S. mutans
strain SJ-r organisms. Rats remained infected with
S. mutans
for 78 days, during which rat molars were sampled for the accumulation of
S. mutans
SJ-r bacteria and total streptococci. Geometric mean levels of
S. mutans
SJ-r accumulation on molar surfaces were significantly lower in antibody-treated rats on days 16 and 78 of experiment 2 and were lower on all but the initial (day 5) swabbing occasions in both experiments. Relative to controls, the extent of molar dental caries measured on day 78 was also significantly decreased. The decrease in molar caries correlated with the amount and duration of antibody administration. This is the first demonstration that passive antibody to
S. mutans
GBP-B can have a protective effect against cariogenic
S. mutans
infection and disease. Furthermore, this decrease in infection and disease did not require continuous antibody administration for the duration of the infection period. This study also indicates that antibody to components putatively involved only in cellular aggregation can have a significant effect on the incorporation of mutans streptococci in dental biofilm.
Isogenic Serotypes ofBorrelia turicataeShow Different Localization in the Brain and Skin of Mice
Authors: Cadavid Diego , Pachner Andrew R. , Estanislao Lydia , Patalapati Ramaprasad , Barbour Alan G. 
Published: July 2002

ABSTRACTMice with severe combined immunodeficiency (scidmice) and infected with the relapsing fever agentBorrelia turicataedevelop manifestations that resemble those of disseminated Lyme disease. We have characterized two isogenic serotypes, A and B, which differ in their variable small proteins (Vsps) and disease manifestations. Serotype A but not serotype B was cultured from the brain during early infection, and serotype B caused more severe arthritis, myocarditis, and vestibular dysfunction than serotype A. Here we compared the localization and number of spirochetes and the severity of inflammation inscidmice, using immunostained and hematoxylin-and-eosin-stained coronal sections of decalcified heads. Spirochetes in the brain localized predominantly to the leptomeninges, and those in peripheral tissues localized mainly to the extracellular matrix. There were significantly more serotype A than B spirochetes in the leptomeninges and more serotype B than A spirochetes in the skin. The first tissue where spirochetes were observed outside the vasculature was the dura mater. Inflammation was more severe in the skin than in the brain. VspA, VspB, and the periplasmic flagellin protein were expressed in all tissues examined. These findings indicate that isogenic but antigenically distinctBorreliaserotypes can have marked differences in their localization in tissues.
Influence of Vector-Encoded Cytokines on Anti-SalmonellaImmunity: Divergent Effects of Interleukin-2 and Tumor Necrosis Factor Alpha
Authors: al-Ramadi Basel K. , Al-Dhaheri Mariam H. , Mustafa Nada , AbouHaidar Mounir , Xu Damu , Liew F. Y. , Lukic Miodrag L. , Fernandez-Cabezudo Maria J. 
Published: July 2002

ABSTRACTAttenuatedSalmonellastrains are of interest as new vaccine candidates and as vectors of cloned genes of other organisms. Attenuated strains expressing specific cytokines were constructed as a means of manipulating the immune response in various disease settings. In the present study, interleukin-2 (IL-2)-expressing (GIDIL2) or tumor necrosis factor alpha (TNF-α)-expressing (GIDTNF) strains were compared with the parent strain (BRD509) for the effect of cytokines on anti-Salmonellaimmunity. Expression of IL-2 resulted in a rapid clearance of the organism soon after vaccination. The reduction in GIDIL2 CFU was 50- to 300-fold higher than that of BRD509 and correlated with a markedly decreased splenomegaly. Furthermore, no evidence for any significant activation, including upregulation of surface markers and production of nitric oxide (NO), was observed in spleens of GIDIL2-injected mice. In contrast, the host response to GIDTNF was marked by an early, strong, splenic cellular influx, but surprisingly, the degree of induced splenomegaly and NO secretion was only 50% of that observed in BRD509-treated mice. Despite this, bacterial colonization of the spleen in GIDTNF-immunized animals was either slightly decreased from or equivalent to that of the BRD509-treated group, suggesting the induction of additional antimicrobial mechanisms by TNF-α. In vivo protection studies demonstrated that, at limiting doses, GIDIL2 was inferior to GIDTNF and BRD509 in its capacity to protect against virulent challenge. At high doses, however, all three strains exhibited equal protective efficacy. These results demonstrate that the immune response against intracellular bacteria can be manipulated by pathogen-expressed cytokines and open the way for further fine tuning of immune responses not only toSalmonellastrains themselves but also to the heterologous gene(s) carried by them.
Vaccination with FimA from
Streptococcus parasanguis
Protects Rats from Endocarditis Caused by Other Viridans Streptococci
Authors: Kitten Todd , Munro Cindy L. , Wang Aijuan , Macrina Francis L. 
Published: July 2002

ABSTRACT

The FimA protein of
Streptococcus parasanguis
is a virulence factor in the rat model of endocarditis, and immunization with FimA protects rats against homologous bacterial challenge. Because FimA-like proteins are widespread among the oral streptococci, the leading cause of native valve endocarditis, we evaluated the ability of this vaccinogen to protect rats when challenged by other streptococcal species. Here we report that FimA vaccination produced antibodies that cross-reacted with and protected against challenge by the oral streptococci
S. mitis
,
S. mutans
, and
S. salivarius
. FimA thus has promise as a vaccinogen to control infective endocarditis caused by oral streptococci.
Porins fromSalmonella entericaSerovar Typhimurium Activate the Transcription Factors Activating Protein 1 and NF-κB through the Raf-1-Mitogen-Activated Protein Kinase Cascade
Authors: Galdiero Massimiliano , Vitiello Mariateresa , Sanzari Emma , D’Isanto Marina , Tortora Annalisa , Longanella Anna , Galdiero Stefania 
Published: July 2002

ABSTRACTIn this study we examined the ability ofSalmonella entericaserovar Typhimurium porins to activate activating protein 1 (AP-1) and nuclear factor κB (NF-κB) through the mitogen-activated protein kinase (MAPK) cascade, and we identified the AP-1-induced protein subunits. Our results demonstrate that these enzymes may participate in cell signaling pathways leading to AP-1 and NF-κB activation following porin stimulation of cells. Raf-1 was phosphorylated in response to the treatment of U937 cells with porins; moreover, the porin-mediated increase in Raf-1 phosphorylation is accompanied by the phosphorylation of MAPK kinase 1/2 (MEK1/2), p38, extracellular-signal-regulated kinase 1/2, and c-Jun N-terminal kinase. We used three different inhibitors of phosphorylation pathways: 2′-amino-3′-methoxyflavone (PD-098059), a selective inhibitor of MEK1 activator and the MAPK cascade; 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), a specific inhibitor of the p38 pathway; and 7β-acetoxy-1α,6β,9α-trihydroxy-8,13-epoxy-labd-14-en-11-one (forskolin), an inhibitor at the level of Raf-1 kinase. PD-098059 pretreatment of cells decreases AP-1 and NF-κB activation by lipopolysaccharide (LPS) but not by porins, and SB203580 pretreatment of cells decreases mainly AP-1 and NF-κB activation by porins; in contrast, forskolin pretreatment of cells does not affect AP-1 and NF-κB activation following either porin or LPS stimulation. Our data suggest that the p38 signaling pathway mainly regulates AP-1 and NF-κB activation in cells treated withS. entericaserovar Typhimurium porins. Antibody electrophoretic mobility shift assays showed that JunD and c-Fos binding is found in cells treated with porins, in cells treated with LPS, and in unstimulated cells. However, by 30 to 60 min of stimulation, a different complex including c-Jun appears in cells treated with porins or LPS, while the Fra-2 subunit is present only after porin stimulation. These data suggest different molecular mechanisms of activation induced by porins or by LPS.
Hydrolysis of Epithelial Junctional Proteins byPorphyromonas gingivalisGingipains
Authors: Katz Jannet , Yang Qiu-Bo , Zhang Ping , Potempa Jan , Travis James , Michalek Suzanne M. , Balkovetz Daniel F. 
Published: July 2002

ABSTRACTPorphyromonas gingivalishas been implicated as an etiologic agent of adult periodontitis. We have previously shown thatP. gingivaliscan degrade the epithelial cell-cell junction complexes, thus suggesting that this bacterium can invade the underlying connective tissues via a paracellular pathway. However, the precise mechanism(s) involved in this process has not been elucidated. The purpose of this study was to determine if the arginine- and lysine-specific gingipains ofP. gingivalis(i.e., HRgpA and RgpB, and Kgp, respectively) were responsible for the degradation of E-cadherin, the cell-cell adhesion protein in the adherens junctions. In addition, we compared the degradative abilities of the whole gingipains HRgpA and Kgp to those of their catalytic domains alone. In these studies, immunoprecipitated E-cadherin as well as monolayers of polarized Madin-Darby canine kidney (MDCK) epithelial cell cultures were incubated with the gingipains and hydrolysis of E-cadherin was assessed by Western blot analysis. Incubation ofP. gingivaliscells with immunoprecipitated E-cadherin resulted in degradation, whereas prior exposure ofP. gingivaliscells to leupeptin and especially acetyl-Leu-Val-Lys-aldehyde (which are arginine- and lysine-specific inhibitors, respectively) reduced this activity. Furthermore, incubation of E-cadherin immunoprecipitates with the different gingipains resulted in an effective and similar hydrolysis of the protein. However, when monolayers of MDCK cells were exposed to the gingipains, Kgp was most effective in hydrolyzing the E-cadherin molecules in the adherens junction. Kgp was more effective than its catalytic domain in degrading E-cadherin at 500 nM but not at a lower concentration (250 nM). These results suggest that the hemagglutinin domain of Kgp plays a role in degradation and that there is a critical threshold concentration for this activity. Taken together, these results provide evidence that the gingipains, especially Kgp, are involved in the degradation of the adherens junction of epithelial cells, which may be important in the invasion of periodontal connective tissue byP. gingivalis.