bluuebunny/crossref_metadata_2025_split huggingface dataset:
Influence of Specimen Age and Use of Different Negative Controls in Determination of Intracytoplasmic Levels of Cytokines after Whole-Blood Culture Assay
Authors: Schultz Christian , Rott Christina , Temming Petra , von Puttkammer Julia , Bucsky Peter 
Published: July 2002

ABSTRACT
Intracytoplasmic detection of cytokines by flow cytometry has become a powerful tool in the characterization of cytokine-producing cells. However, it is not known to what extent specimen age and the use of various negative controls may influence the amount of cytokine-positive cells. We therefore compared different times of storage and the use of several negative controls in the determination of intracytoplasmic levels of cytokines. There was a substantial decline of interleukin-2- and gamma interferon-positive lymphocytes after 20 h and especially after 48 h of storage. The precision of intracytoplasmic interleukin-6 determination decreases after long-term storage compared to 2 h of storage, whereas the amount of interleukin-8-positive monocytes remained rather stable. Therefore, we recommend performing the analysis as fast as possible after the blood sample is drawn. Under consideration of isotype-matched antibodies and nonstimulated cells as negative controls instead of the purified antibody-blocking control, strikingly higher amounts of interleukin-2-, gamma interferon, interleukin-6-, and interleukin-8-positive cells were found. For a meaningful interpretation of data these differences have to be kept in mind. Further studies should evaluate the exact specificity of these controls.
Predicting Kala-Azar Disease Manifestations in Asymptomatic Patients with LatentLeishmania donovaniInfection by Detection of Antibody against Recombinant K39 Antigen
Authors: Singh Sarman , Kumari Veena , Singh Niti 
Published: July 2002

ABSTRACTClinically visceral leishmaniasis is suspected in only a fraction of infected persons, as the majority of these may not have clinical manifestations and remain asymptomatic. There is scanty information on diagnosing latent infections and predicting disease in asymptomatic persons. We therefore carried out a study on asymptomatic contacts of patients with visceral leishmaniasis and post-kala-azar dermal leishmaniasis by using methods for detection of antibody to recombinant K39 (rK39) antigen. A total of 240 patients with leishmaniasis and 150 asymptomatic contacts were tested for anti-rK39 immunoglobulin G (IgG) and IgA antibodies. Fifty-five asymptomatic persons were found to be seropositive. These individuals were monitored every 3 months for 1 year. On follow-up, 43.9% of the asymptomatic seropositive contacts developed kala-azar within the first 3 months, and a cumulative total of 69% developed kala-azar within 1 year. The rest remained asymptomatic and self-healed the infection. The sensitivity and specificity of rK39 enzyme-linked immunosorbent assay (ELISA) and dipstick tests were 100%, while an in-house-developed latex agglutination test had 80% sensitivity. The antibody profile showed that the IgG anti-rK39 antibodies reached a titer of up to 10−6within 6 months of infection, started declining thereafter, and completely disappeared in 2 to 3 years in successfully treated cases. Significant titers of IgA antibodies were detectable a little earlier than those of IgG antibodies and were undetectable after 6 months. The study showed that mass screening of family members and contacts by using anti-rK39 ELISA could be a highly reliable tool for early diagnosis and to plan prophylactic treatment of latently infected asymptomatic carriers to eradicate kala-azar.
Leishmaniasis: Current Status of Vaccine Development
Authors: Handman Emanuela 
Published: July 2002

SUMMARYLeishmaniae are obligatory intracellular protozoa in mononuclear phagocytes. They cause a spectrum of diseases, ranging in severity from spontaneously healing skin lesions to fatal visceral disease. Worldwide, there are 2 million new cases each year and 1/10 of the world's population is at risk of infection. To date, there are no vaccines against leishmaniasis and control measures rely on chemotherapy to alleviate disease and on vector control to reduce transmission. However, a major vaccine development program aimed initially at cutaneous leishmaniasis is under way. Studies in animal models and humans are evaluating the potential of genetically modified live attenuated vaccines, as well as a variety of recombinant antigens or the DNA encoding them. The program also focuses on new adjuvants, including cytokines, and delivery systems to target the T helper type 1 immune responses required for the elimination of this intracellular organism. The availability, in the near future, of the DNA sequences of the human and Leishmania genomes will extend the vaccine program. New vaccine candidates such as parasite virulence factors will be identified. Host susceptibility genes will be mapped to allow the vaccine to be targeted to the population most in need of protection.
Phase Variation in H Type I and Lewis a Epitopes of
Helicobacter pylori
Lipopolysaccharide
Authors: Appelmelk Ben J. , Martino M. Celeste , Veenhof Eveline , Monteiro Mario A. , Maaskant Janneke J. , Negrini Riccardo , Lindh Frank , Perry Malcolm , Del Giudice Giuseppe , Vandenbroucke-Grauls Christina M. J. E. 
Published: July 2002

ABSTRACT

Helicobacter pylori
NCTC 11637 lipopolysaccharide (LPS) expresses the human blood group antigens Lewis x (Le
x
), Le
y
, and H type I. In this report, we demonstrate that the H type I epitope displays high-frequency phase variation. One variant expressed Le
x
and Le
y
and no H type I as determined by serology; this switch was reversible. Insertional mutagenesis in NCTC 11637 of JHP563 (a poly(C) tract containing an open reading frame homologous to glycosyltransferases) yielded a transformant with a serotype similar to the phase variant. Structural analysis of the NCTC 11637 LPS confirmed the loss of the H type I epitope. Sequencing of JHP563 in strains NCTC 11637, an H type I-negative variant, and an H type I-positive switchback variant showed a C14 (gene on), C13 (gene off), and C14 tract, respectively. Inactivation of strain G27, which expresses Le
x
, Le
y
, H type I, and Le
a
, yielded a transformant that expressed Le
x
and Le
y
. We conclude that JHP563 encodes a β3-galactosyltransferase involved in the biosynthesis of H type I and Le
a
and that phase variation in H type I is due to C-tract changes in this gene. A second H type I-negative variant (variant 3a) expressed Le
x
and Le
a
and had lost both H type I and Le
y
expression. Inactivation of HP093-HP094 resulted in a transformant expressing Le
x
and lacking Le
y
and H type I. Structural analysis of a mutant LPS confirmed the serological data. We conclude that the HP093-HP094 α2-fucosyltransferase (α2-FucT) gene product is involved in the biosynthesis of both Le
y
and Le
x
. Finally, we inactivated HP0379 in strain 3a. The transformant had lost both Le
x
and Le
a
expression, which demonstrates that the HP0379 gene product is both an α3- and an α4-FucT. Our data provide understanding at the molecular level of how
H. pylori
is able to diversify in the host, a requirement likely essential for successful colonization and transmission.
ClpC ATPase Is Required for Cell Adhesion and Invasion of
Listeria monocytogenes
Authors: Nair Shamila , Milohanic Eliane , Berche Patrick 
Published: July 2002

ABSTRACT

We studied the role of two members of the 100-kDa heat shock protein family, the ClpC and ClpE ATPases, in cell adhesion and invasion of the intracellular pathogen
Listeria monocytogenes
. During the early phase of infection, a
clpC
mutant failed to disseminate to hepatocytes in the livers of infected mice whereas the invasive capacity of a
clpE
mutant remained unchanged. This was confirmed by a confocal microscopy study on infected cultured hepatocyte and epithelial cell lines, showing a strong reduction of cell invasion only by the
clpC
mutant. Western blot analysis with specific antisera showed that the absence of ClpC, but not that of ClpE, reduced expression of the virulence factors InlA, InlB, and ActA. ClpC-dependent modulation of these factors occurs at the transcriptional level with a reduction in the transcription of
inlA
,
inlB
, and
actA
in the
clpC
mutant, in contrast to the
clpE
mutant. This work provides the first evidence that, in addition to promoting escape from the phagosomes, ClpC is required for adhesion and invasion and modulates the expression of InlA, InlB, and ActA, further supporting the major role of the Clp chaperones in the virulence of intracellular pathogens.
Effect of
Chlamydia trachomatis
Infection on Atherosclerosis in Apolipoprotein E-Deficient Mice
Authors: Blessing Erwin , Nagano Sanae , Campbell Lee Ann , Rosenfeld Michael E. , Kuo Cho-chou 
Published: July 2002

ABSTRACT

We have previously demonstrated that
Chlamydia pneumoniae
accelerates plaque formation in apolipoprotein E-deficient (ApoE
−/−
) mice following intranasal inoculations. In this study, we evaluated the effect of respiratory tract infection with
Chlamydia trachomatis
on the progression of atherosclerosis in ApoE
−/−
mice. The study showed that in contrast to infection with
Chlamydia pneumoniae
, infection of the lung and aorta with
C. trachomatis
was mild and transient and did not significantly accelerate plaque development.
Requirement for CD4+T Lymphocytes in Host Resistance againstCryptococcus neoformansin the Central Nervous System of Immunized Mice
Authors: Buchanan Kent L. , Doyle Hester A. 
Published: July 2002

ABSTRACTThe importance of cell-mediated immunity (CMI) and CD4+T lymphocytes in host resistance againstCryptococcus neoformansis well documented and is exemplified by the high susceptibility to progressive infection with this pathogen of AIDS patients with reduced CD4+T-cell numbers. Although much has been learned about the role of CMI in the clearance ofC. neoformansfrom the lungs and other internal organs, less is known about the protective mechanisms in the brain, the organ most frequently involved with a fatal outcome of cryptococcosis. We hypothesized that host resistance mechanisms againstC. neoformansin the central nervous system (CNS) were similar to those outside the CNS (i.e., gamma interferon [IFN-γ], CD4+T cells, and others). To test this hypothesis, we used a murine model of cryptococcal meningitis whereby cryptococci are introduced directly into the CNS. In experiments where mice were immunized to mount an anticryptococcal CMI response, our results indicate that immunization induced protective mechanisms that could be detected in the CNS by inhibition of the growth of viable yeast cells. Flow cytometric analyses of leukocytes in brain and spinal cord homogenates revealed that T lymphocytes, macrophages, and neutrophils accumulated inC. neoformans-infected brains of immune mice. In vivo depletion of CD4+T cells, but not CD8+T cells, resulted in significantly reduced leukocyte accumulation in the brains of immune mice. Furthermore, depletion of CD4+T cells or neutralization of IFN-γ exacerbated CNS infection in immune mice, suggesting a critical role for CMI mechanisms in acquired protection in the CNS.
Febrile Core Temperature Is Essential for Optimal Host Defense in Bacterial Peritonitis
Authors: Jiang Qinqqi , Cross Alan S. , Singh Ishwar S. , Chen T. Timothy , Viscardi Rose M. , Hasday Jeffrey D. 
Published: July 2002

ABSTRACTFever, a nonspecific acute-phase response, has been associated with improved survival and shortened disease duration in infections, but the mechanisms of these beneficial responses are poorly understood. We previously reported that increasing core temperature of bacterial endotoxin (LPS)-challenged mice to the normal febrile range modified expression of tumor necrosis factor alpha (TNF-α), interleukin 1β (IL-1β), and IL-6, three cytokines critical to mounting an initial defense against microbial pathogens, but survival was not improved in the warmer animals. We speculated that our inability to show a survival benefit of optimized cytokine expression in the warmer animals reflected our use of LPS, a nonreplicating agonist, rather than an infection with viable pathogens. The objective of this study was to determine if increasing murine core temperature altered cytokine expression and improved survival in an experimental bacterial peritonitis model. We showed that housing mice at 35.5°C rather than 23°C increased core temperature from 36.5 to 37.5°C to 39.2 to 39.7°C, suppressed plasma TNF-α expression for the initial 48 h, delayed gamma interferon expression, improved survival, and reduced the bacterial load in mice infected withKlebsiella pneumoniaeperitonitis. We showed that the reduced bacterial load was not caused by a direct effect on bacterial proliferation and probably reflected enhanced host defense. These data suggest that the increase in core temperature that occurs during bacterial infections is essential for optimal antimicrobial host defense.
Analysis of the F Antigen-Specific
papA
Alleles of Extraintestinal Pathogenic
Escherichia coli
Using a Novel Multiplex PCR-Based Assay
Authors: Johnson James R. , Stell Adam L. , Scheutz Flemming , O'Bryan Timothy T. , Russo Thomas A. , Carlino Ulrike B. , Fasching Claudine , Kavle Justine , Van Dijk Linda , Gaastra Wim 
Published: July 2002

ABSTRACT

Polymorphisms in PapA, the major structural subunit and antigenic determinant of P fimbriae of extraintestinal pathogenic
Escherichia coli
, are of considerable epidemiological, phylogenetic, and immunotherapeutic importance. However, to date, no method other than DNA sequencing has been generally available for their detection. In the present study, we developed and rigorously validated a novel PCR-based assay for the 11 recognized variants of
papA
and then used the new assay to assess the prevalence, phylogenetic distribution, and bacteriological associations of the
papA
alleles among 75
E. coli
isolates from patients with urosepsis. In comparison with conventional F serotyping, the assay was extremely sensitive and specific, evidence that
papA
sequences are highly conserved within each of the traditionally recognized F serotypes despite the diversity observed among F types. In certain strains, the assay detected serologically occult copies of
papA
, of which some were shown to represent false-negative serological results and others were shown to represent the presence of nonfunctional
pap
fragments. Among the urosepsis isolates, the assay revealed considerable segregation of
papA
alleles according to O:K:H serotype, consistent with vertical transmission within clones, but with exceptions which strongly suggested horizontal transfer of
papA
alleles between lineages. Sequencing of
papA
from two strains that were
papA
positive by probe and PCR but F negative in the new PCR assay led to the discovery of two novel
papA
variants, one of which was actually more prevalent among the urosepsis isolates than were several of the known
papA
alleles. These findings provide novel insights into the
papA
alleles of extraintestinal pathogenic
E. coli
and indicate that the F PCR assay represents a versatile new molecular tool for epidemiological and phylogenetic investigations which should make rapid, specific detection of
papA
alleles available to any laboratory with PCR capability.
Effective Preexposure Tuberculosis Vaccines Fail To Protect When They Are Given in an Immunotherapeutic Mode
Authors: Turner Joanne , Rhoades Elizabeth R. , Keen Marc , Belisle John T. , Frank Anthony A. , Orme Ian M. 
Published: July 2002

ABSTRACTTwo vaccine formulations previously shown to induce protective immunity in mice and prevention of long-term necrosis in guinea pigs were tested as potential immunotherapeutic vaccines in mice earlier infected by aerosol withMycobacterium tuberculosis. Neither vaccine had any effect on the course of the infection in the lungs, but both reduced the bacterial load in the spleen. Similarly, inoculation withMycobacterium bovisBCG had no effect whatsoever and, if given more than once, appeared to induce an increasingly severe pyogranulomatous response in the lungs of these mice.
Antibodies to Tyvelose Exhibit Multiple Modes of Interference with the Epithelial Niche ofTrichinella spiralis
Authors: McVay Catherine S. , Bracken Peter , Gagliardo Lucille F. , Appleton Judith 
Published: July 2002

ABSTRACTInfection with the parasitic nematodeTrichinella spiralisis initiated when the L1 larva invades host intestinal epithelial cells. Monoclonal antibodies specific for glycans on the larval surface and secreted glycoproteins protect the intestine against infection. Protective antibodies recognize tyvelose which caps the target glycan. In this study, we used an in vitro model of invasion to further examine the mechanism(s) by which tyvelose-specific antibodies protect epithelial cells againstT. spiralis. Using cell lines that vary in susceptibility to invasion, we confirmed and clarified the results of our in vivo studies by documenting three modes of interference: exclusion of larvae from cells, encumbrance of larvae as they migrated within epithelial monolayers, and inhibition of parasite development. Excluded larvae bear cephalic caps (C. S. McVay et al., Infect. Immun. 66:1941–1945, 1998) of immune complexes that may physically block invasion or may interfere with sensory reception. Monovalent Fab fragments prepared from a tyvelose-specific antibody also excluded larvae from cells, demonstrating that antibody binding can inhibit the parasite in the absence of antigen aggregation and cap formation. In contrast, encumbered larvae caused extensive damage to the monolayer yet were not successful in establishing a niche, as reflected by their failure to molt. These results show that antibodies to tyvelose exhibit multiple modes of inhibitory activity, further implicating tyvelose-bearing glycoproteins as mediators of invasion and niche establishment byT. spiralis.
mkp-1
Encoding Mitogen-Activated Protein Kinase Phosphatase 1, a Verotoxin 1 Responsive Gene, Detected by Differential Display Reverse Transcription-PCR in Caco-2 Cells
Authors: Kojima Shihoko , Yanagihara Itaru , Kono Gengo , Sugahara Tomomi , Nasu Hatsumi , Kijima Mika , Hattori Akiko , Kodama Toshio , Nagayama Ken-Ichi , Honda Takeshi 
Published: July 2002

ABSTRACT

The major cytotoxic effect of the verotoxins (VTs) produced by strains of VT-producing
Escherichia coli
is the inhibition of host-cell protein synthesis, but VTs are also suspected to play a role in apoptotic cell signaling and cytokine release. Four differentially expressed genes, including
mkp-1
(encoding mitogen-activated protein kinase phospatase 1), were detected by differential display reverse transcription-PCR (DD RT-PCR) stimulated by VT1 in Caco-2 cells. Northern blot analysis showed the induction of
mkp-1
mRNA 6 h after VT1 stimulation. Neither mutant VT1 (mutVT1), harboring two mutations in the A subunit (E167Q-R170L), nor cycloheximide induced
mkp-1
mRNA, but
mkp-1
mRNA was detected with both wild-type VT1 (wtVT1) and anisomycin, a 28S rRNA inhibitor. Therefore, we concluded that the A subunit of VT1 was essential for
mkp-1
induction. Increased amounts of phosphorylated c-Jun protein were also found with wtVT1 and anisomycin. Although the precise mechanism of induction of MKP-1 is unknown, we hypothesized that 28S rRNA not only was a sensor for ribotoxic stress, but also was involved in the signal cascade of MKP-1. This is the first report of detection by DD RT-PCR of cellular genes induced by bacterial toxins.
Intracellular Growth ofLegionella pneumophilainDictyostelium discoideum, a System for Genetic Analysis of Host-Pathogen Interactions
Authors: Solomon Jonathan M. , Rupper Adam , Cardelli James A. , Isberg Ralph R. 
Published: July 2002

ABSTRACTConditions were established in whichLegionella pneumophila, an intracellular bacterial pathogen, could replicate within the unicellular organismDictyostelium discoideum. By several criteria,L. pneumophilagrew by the same mechanism withinD. discoideumas it does in amoebae and macrophages. Bacteria grew within membrane-bound vesicles associated with rough endoplasmic reticulum, andL. pneumophila dot/icmmutants, blocked for growth in macrophages and amoebae, also did not grow inD. discoideum. InternalizedL. pneumophilaavoided degradation byD. discoideumand showed evidence of reduced fusion with endocytic compartments. The ability ofL. pneumophilato grow withinD. discoideumdepended on the growth state of the cells.D. discoideumgrown as adherent monolayers was susceptible toL. pneumophilainfection and to contact-dependent cytotoxicity during high-multiplicity infections, whereasD. discoideumgrown in suspension was relatively resistant to cytotoxicity and did not support intracellular growth. Some knownD. discoideummutants were examined for their effect on growth ofL. pneumophila. The coronin mutant and themyoA/Bdouble myosin I mutant were more permissive than wild-type strains for intracellular growth. Growth ofL. pneumophilain a Gβmutant was slightly reduced compared to the parent strain. This work demonstrates the usefulness of theL. pneumophila-D. discoideumsystem for genetic analysis of host-pathogen interactions.
Tumor Necrosis Factor Alpha-Mediated Toxic Shock inTrypanosoma cruzi-Infected Interleukin 10-Deficient Mice
Authors: Hölscher Christoph , Mohrs Markus , Dai Wen Juan , Köhler Gabriele , Ryffel Bernhard , Schaub Günter A. , Mossmann Horst , Brombacher Frank 
Published: July 2002

ABSTRACTUsing interleukin-10 (IL-10)-deficient (IL-10−/−) mice, previous studies revealed a pathological immune response after infection withTrypanosoma cruzithat is associated with CD4+T cells and overproduction of proinflammatory cytokines. In this study we further investigate the pathology and potential mediators for the mortality in infected animals.T. cruzi-infected IL-10−/−mice showed reduced parasitemia accompanied by increased systemic release of gamma interferon (IFN-γ), IL-12, and reactive nitrogen intermediates and overproduction of tumor necrosis factor alpha (TNF-α). Despite this early resistance, IL-10−/−mice died within the third week of infection, whereas all control mice survived acute infection. The clinical manifestation with weight loss, hypothermia, hypoglycemia, hyperkalemia, and increased liver-derived enzymes in the blood together with hepatic necrosis and intravascular coagulation in moribund mice indicated a toxic shock-like syndrome, possibly mediated by the systemic TNF-α overproduction. Indeed, high production of systemic TNF-α significantly correlated with mortality, and moribund mice died with critically high TNF-α concentrations in the blood. Consequent treatment with anti-TNF-α antiserum attenuated pathological changes inT. cruzi-infected IL-10−/−mice and significantly prolonged survival; the mice died during the fourth week postinfection, again with a striking correlation between regaining high systemic TNF-α concentrations and the time of death. Since elevated serum IL-12 and IFN-γ concentrations were not affected by the administration of antiserum, these studies suggest that TNF-α is the direct mediator of this toxic shock syndrome. In conclusion, induction of endogenous IL-10 during experimentally induced Chagas' disease seems to be crucial for counterregulating an overshooting proinflammatory cytokine response resulting in TNF-α-mediated toxic shock.