bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Vivo Distribution ofHelicobacter felisin the Gastric Mucus of the Mouse: Experimental Method and Results
Authors: Schreiber Sören , Stüben Manuela , Josenhans Christine , Scheid Peter , Suerbaum Sebastian 
Published: December 2019

ABSTRACTWe describe a method that permits the collection of very small samples (2 nl) from precisely defined positions within the gastric mucus of anesthetized mice. This method was used to study the in vivo local distribution of bacteria within the mucus ofHelicobacter felis-infected mice. A total of 200 samples from 40 mice were analyzed. Each sample was microscopically analyzed, within less than 1 min, as a native preparation. To avoid changes in bacterial location within the mucus after collection and to improve the counting accuracy, bacterial motility was blocked by adjusting the pH inside the collecting pipette to 4.5. The mucus in a collected sample was subdivided into three layers, an epithelial layer (the first 25 μm of mucus from the tissue-mucus interface), a luminal layer (the last 25 μm to the mucus-lumen interface), and the remaining central mucus layer. The volume of the analyzed segments in the sample was between 4 and 9 pl. The concentration of bacteria inside the epithelial mucus layer was 3,400 per nl, but it was only 50 per nl inside the central mucus layer. The mean distance ofH. felisto the epithelial surface was 16 μm. A total of 75% of allH. felisbacteria resided in the mucus zone between 5 and 20 μm from the tissue surface, with no bacteria closer than 5 μm to the epithelial surface. This method permits the study of factors determining the density of colonization and distribution of bacteria along chemical gradients with a high precision.
Immune Responses Induced by Gene Gun or Intramuscular Injection of DNA Vaccines That Express Immunogenic Regions of the Serine Repeat Antigen fromPlasmodium falciparum
Authors: Belperron Alexia A. , Feltquate David , Fox Barbara A. , Horii Toshihiro , Bzik David J. 
Published: December 2019

ABSTRACTThe liver- and blood-stage-expressed serine repeat antigen (SERA) ofPlasmodium falciparumis a candidate protein for a human malaria vaccine. We compared the immune responses induced in mice immunized with SERA-expressing plasmid DNA vaccines delivered by intramuscular (i.m.) injection or delivered intradermally by Gene Gun immunization. Mice were immunized with a pcdna3 plasmid encoding the entire 47-kDa domain of SERA (amino acids 17 to 382) or the N-terminal domain (amino acids 17 to 110) of SERA. Minimal antibody responses were detected following DNA vaccination with the N-terminal domain of SERA, suggesting that the N-terminal domain alone is not highly immunogenic by this route of vaccine delivery. Immunization of mice by Gene Gun delivery of the 47-kDa domain of SERA elicited a significantly higher serum antibody titer to the antigen than immunization of mice by i.m. injection with the same plasmid did. The predominant isotype subclass of the antibodies elicited to the SERA protein following i.m. and Gene Gun immunizations with SERA plasmid DNA was immunoglobulin G1. Coimmunization of mice with SERA plasmid DNA and a plasmid expressing the hepatitis B surface antigen (pCMV-s) by the i.m. route resulted in higher anti-SERA titers than those generated in mice immunized with the SERA DNA plasmid alone. Vaccination with DNA may provide a viable alternative or may be used in conjunction with protein-based subunit vaccines to maximize the efficacy of a human malaria vaccine that includes immunogenic regions of the SERA protein.
Induction of Cytokine Synthesis by Flagella from Gram-Negative Bacteria May Be Dependent on the Activation or Differentiation State of Human Monocytes
Authors: Ciacci-Woolwine Federica , McDermott Patrick F. , Mizel Steven B. 
Published: December 2019

ABSTRACTWe have previously demonstrated that salmonellae, but notEscherichia coliorYersinia enterocolitica, stimulates tumor necrosis factor alpha (TNFα) production in the human promonocytic cell line U38. Subsequent analysis revealed that the TNFα-inducing activity of salmonellae was associated with flagellin, a major component of flagella from gram-negative bacteria. In the present study, we have explored the basis for the apparent specificity of action ofSalmonellaflagella on TNFα expression in U38 cells and have extended this analysis to normal human peripheral blood mononuclear cells (PBMC). Flagella from the enteropathogenicE. colistrain E2348/69,Y. enterocoliticaJB580, andPseudomonas aeruginosaPAO1, which did not induce significant levels of TNFα production in U38 cells, were as potent asSalmonellaflagella in terms of TNFα and interleukin 1β activation in PBMC. However, TNFα production in U38 cells was greatly enhanced when these cells were stimulated with flagella fromE. coli,Y. enterocolitica, andP. aeruginosain the presence of a costimulant, phorbol 13-myristate acetate. These findings are consistent with the hypothesis that the activation or differentiation state of a monocyte may have a substantial effect on the cell’s responsiveness to flagellum stimulation of cytokine synthesis. Furthermore, these results indicate that cytokine induction in monocytes may be a general property of flagella from gram-negative bacteria.
P2Z-Independent and P2Z Receptor-Mediated Macrophage Killing byPseudomonas aeruginosaIsolated from Cystic Fibrosis Patients
Authors: Zaborina Olga , Misra Namita , Kostal Jan , Kamath Shilpa , Kapatral Vinayak , El-Idrissi M. El-Azami , Prabhakar B. S. , Chakrabarty A. M. 
Published: December 2019

ABSTRACTWe demonstrate that a mucoid, alginate-producing strain ofPseudomonas aeruginosaisolated from the lungs of a cystic fibrosis (CF) patient secretes multiple enzymes with nucleoside diphosphate kinase (Ndk), ATPase, adenylate kinase, 5′-nucleotidase, and ATP-modifying enzymatic activities. The secretion is triggered at high cell density and in complex media but is greatly reduced when the mucoid cells are grown in mineral salts media or in presence of 5.0 mM Ca2+or Mg2+. Interestingly, the secretion is triggered primarily in the mucoid CF isolate of strain 8821M (or in strain FRD1) but not in a nonmucoid laboratory strain, PAO1. The purified secreted Ndk shows 100% match in its N-terminal amino acid sequence with that of purified intracellular Ndk and demonstrates similar enzymatic properties. The N-terminal sequence of the purified ATPase isolated from anndkknockout mutant shows its identity with that of the heat shock chaperonin Hsp60. During fractionation, the flowthrough fraction from a Mono Q column demonstrates the presence of 5′-nucleotidase, adenylate kinase, and a putative ATP reductase activity. These fractions demonstrate high cytotoxic activities for murine peritoneal primary macrophages which can be further stimulated in the presence of ATP or inhibited by pretreatment of macrophages with oxidized ATP (oATP). The cytotoxicity associated with ATP-induced stimulation is believed to be due to activation of macrophage surface-associated P2Z (P2X7) receptors, which are one of the purinergic receptors responsible for pore formation on macrophage membrane. Blocking of these receptors by pretreatment with oATP blocks ATP-induced macrophage cell death. Thus mucoidP. aeruginosacells elaborate enzymes that modulate the external ATP levels of macrophages, thereby modulating macrophage cell death through P2Z receptor activation. Evidence for the presence of secreted cytotoxic agents that act independently of P2Z receptor activation is also presented.
Tyrosine Phosphatase SHP-1 Is Involved in CD66-Mediated Phagocytosis of Opa52-ExpressingNeisseria gonorrhoeae
Authors: Hauck Christof R. , Gulbins Erich , Lang Florian , Meyer Thomas F. 
Published: December 2019

ABSTRACTOpa proteins ofNeisseria gonorrhoeaebind to CD66 receptors on human phagocytes, thereby inducing efficient uptake of the bacteria in the absence of opsonins. The interaction of Opa proteins and CD66 receptors leads to activation of Src family tyrosine kinases, a process that is of critical importance for the efficient, CD66-mediated internalization. Here we show that during Opa-mediated stimulation of CD66 the activity of the host cell tyrosine phosphatase SHP-1 is strongly downregulated, concomitant with increases in the tyrosine phosphorylation of several cellular proteins. Since the SHP-1 tyrosine phosphorylation level itself is influenced by Opa-induced events, this phosphatase comprises an important regulatory checkpoint of the pathogen-triggered signaling cascade in human phagocytes.
Rational Live Oral Carrier Vaccine Design by Mutating Virulence-Associated Genes of
Yersinia enterocolitica
Authors: Igwe Emeka I. , Rüssmann Holger , Roggenkamp Andreas , Noll Annette , Autenrieth Ingo B. , Heesemann J. 
Published: December 2019

ABSTRACT

Three different
Yersinia enterocolitica
serotype O8 strains harboring mutations in virulence-associated genes coding for
Yersinia
adhesin A (YadA), Mn-cofactored superoxide dismutase (SodA), and high-molecular-weight protein 1 were analyzed for their ability to colonize and persist in tissues after orogastric immunization of C57BL/6 mice. We demonstrated that all three
Yersinia
mutant strains were markedly impaired in their ability to disseminate into the spleens and livers of immunized mice but were able to colonize the Peyer’s patches for at least 12 days, resulting in the induction of significant antibody titers against
Yersinia
outer proteins (Yops) and in the priming of
Yersinia
antigen-specific CD4
+
Th1 cells isolated from spleens. The high level of attenuation did not diminish the immunogenic properties of the mutant strains. In fact, mice immunized with a single oral dose of any of the mutant strains were protected against a lethal oral-challenge infection with wild-type
Y. enterocolitica
. Moreover, adoptive transfer of
Yersinia
-specific antibodies from sera of mice immunized with the mutant WAP-314
sodA
revealed that this protection could be mediated by
Yersinia
-specific immunoglobulins.
Differentiation of Monocytes to Macrophages Primes Cells for Lipopolysaccharide Stimulation via Accumulation of Cytoplasmic Nuclear Factor κB
Authors: Takashiba Shogo , Van Dyke Thomas E. , Amar Salomon , Murayama Yoji , Soskolne Aubrey W. , Shapira Lior 
Published: December 2019

ABSTRACTDuring infection, circulating blood monocytes migrate from the vasculature to the extravascular compartments where they mature into tissue macrophages. The maturation process prepares the cell to actively participate in the inflammatory and the immune responses, and many transcription factors have been found to be involved. Here we report on a novel role for nuclear factor κB (NF-κB) in this process. Its accumulation in the cytoplasm of differentiated macrophages is responsible for the enhanced ability of the cell to respond to lipopolysaccharide (LPS) stimulation, as determined by tumor necrosis factor alpha (TNF-α) secretion. Differentiation of the human monocytic cell line THP-1 into macrophage-like cells was induced by exposure of the cells to phorbol myristate acetate. DNA-bindable NF-κB was not detected in the cytoplasm of undifferentiated THP-1 cells but accumulated in the cytoplasm of the cells following differentiation. No TNF-α was detected in the media of resting differentiated and nondifferentiated THP-1 cells. Stimulation with LPS of differentiated cells induced the production of higher levels of TNF-α than stimulation of nondifferentiated cells. This hyperresponsiveness to LPS was found in the mRNA and secreted TNF-α levels. Furthermore, stimulation with LPS induced the translocation of NF-κB from the cytoplasm into the nucleus. This translocation process was more rapid in the differentiated cells than in the nondifferentiated cells, and the resultant accumulated levels of NF-κB in the nucleus were higher. The DNA-bindable NF-κB was identified as a heterodimer of p65 and p50. The results suggest that NF-κB accumulation in the cytoplasm during maturation of monocytes to macrophages primes the cells for enhanced responsiveness to LPS and results in the rapid secretion of inflammatory mediators, such as TNF-α, by mature macrophages following LPS challenge.
Endothelial Adhesion Molecule Expression and Its Inhibition by Recombinant Bactericidal/Permeability-Increasing Protein Are Influenced by the Capsulation and Lipooligosaccharide Structure ofNeisseria meningitidis
Authors: Dixon Garth L. J. , Heyderman Robert S. , Kotovicz Karolena , Jack Dominic L. , Andersen Svein R. , Vogel Ulrich , Frosch Matthias , Klein Nigel 
Published: December 2019

ABSTRACTVascular endothelial injury is responsible for many of the clinical manifestations of severe meningococcal disease. Binding and migration of activated host inflammatory cells is a central process in vascular damage. The expression and function of adhesion molecules regulate interactions between leukocytes and endothelial cells. Little is known about how meningococci directly influence these receptors. In this study we have explored the effect ofNeisseria meningitidison endothelial adhesion molecule expression and found this organism to be a potent inducer of the adhesion molecules CD62E, ICAM-1, and VCAM-1. Exposure of endothelium to a serogroup B strain ofNeisseria meningitidis, B1940, and a range of isogenic mutants revealed that lipooligosaccharide (LOS) structure and capsulation influence the expression of adhesion molecules. Following only a brief exposure (15 min) to the bacteria, there were large differences in the capacity of the different mutants to induce vascular cell adhesion molecules, with the unencapsulated and truncated LOS strains being most potent (P< 0.05). Furthermore, the pattern of cell adhesion molecule expression was different with purified endotoxin from that with intact bacteria. Meningococci were more potent stimuli of CD62E expression than was endotoxin, whereas endotoxin was at least as effective as meningococci in inducing ICAM-1 and VCAM-1. The effect of bactericidal/permeability increasing protein (rBPI21), an antibacterial molecule with antiendotoxin properties, was also dependent on LOS structure. The strains which possessed a truncated or nonsialylated LOS, whether capsulated or not, were more sensitive to the inhibitory effects of rBPI21. These findings could have important implications for the use of antiendotoxin therapy in meningococcal disease.
Monoclonal Antibody to Shiga Toxin 2 Which Blocks Receptor Binding and Neutralizes Cytotoxicity
Authors: Nakao Hiroshi , Kiyokawa Nobutaka , Fujimoto Junichiro , Yamasaki Shinji , Takeda Tae 
Published: December 2019

ABSTRACT

A monoclonal antibody (MAb) was raised against Shiga toxin 2 (Stx2) of
Escherichia coli
O157:H7. MAb VTm1.1 belonged to the immunoglobulin G1 subclass and had a κ light chain, and it could neutralize the cytotoxic activity of Stx2 and variants derived from patient strains but not that of variants derived from animals. MAb VTm1.1 was shown to bind to the B subunit of these neutralized Stx2s by Western blotting. Comparison of B-subunit amino acid sequences and reactivities to these Stxs suggested six amino acids (Ser30, Ser53, Glu56, Gln65, Asn68, and Asp69) that were candidates for the MAb VTm1.1 epitope. Consequently, five Stx2 mutants (S30N, S53N, E56H, Q65K, and N68Ter) were prepared by site-directed mutagenesis to determine which residue is essential for the epitope. All of these mutants showed cytotoxicity almost equal to that of the wild-type Stx2. Of the five Stx2 mutants, only E56H could not be neutralized by MAb VTm1.1. Western blot analysis also showed that MAb VTm1.1 could not bind to the E56H B subunit. These results indicated that Glu56 is an important residue recognized by MAb VTm1.1. Immunofluorescence analysis further indicated that MAb VTm1.1 inhibits the binding of Stx2 to its receptors. MAb VTm1.1 could be a useful therapeutic agent for Shiga toxin-producing
E. coli
infection.
Emergence of
Anaplasma marginale
Antigenic Variants during Persistent Rickettsemia
Authors: French Dorothy M. , Brown Wendy C. , Palmer Guy H. 
Published: December 2019

ABSTRACT

Anaplasma marginale
is an ehrlichial pathogen of cattle, in the order
Rickettsiales
, that establishes persistent cyclic rickettsemia in the infected host. Within each rickettsemic cycle,
A. marginale
expressing antigenically variant major surface protein 2 (MSP2) emerge. By cloning 17 full-length
msp2
transcripts expressed during cyclic rickettsemia, we determined that emergent variants have a single, central hypervariable region encoding variant B-cell epitopes. The N- and C-terminal regions are highly conserved among the expressed
A. marginale
variants, and similar sequences define the MSP2 homologues in the agent of human granulocytic ehrlichiosis (HGE). This is in contrast to the MSP2 homologues in ehrlichial genogroup I pathogens,
Ehrlichia chaffeensis
,
Ehrlichia canis
, and
Cowdria ruminantium
, that have multiple hypervariable regions. By defining the variable and conserved regions, we were able to show that the single hypervariable region of
A. marginale
MSP2 encodes epitopes that are immunogenic and induce variant-specific antibody responses during persistent infection. These findings demonstrate that the MSP2 structural variants that emerge during each cycle of persistent rickettsemia are true antigenic variants, consistent with MSP2 antigenic variation as a mechanism of
A. marginale
persistence.
Targeted Salivary Gland Immunization with Plasmid DNA Elicits Specific Salivary Immunoglobulin A and G Antibodies and Serum Immunoglobulin G Antibodies in Mice
Authors: Kawabata Shigetada , Terao Yutaka , Fujiwara Taku , Nakagawa Ichiro , Hamada Shigeyuki 
Published: December 2019

ABSTRACTFor the development of vaccines against oral and pharyngeal pathogens invading the mucosal epithelia, both secretory and serum immunoglobulin A (IgA) and IgG antibodies and cytotoxic T lymphocytes (CTL) have been induced. We used a novel approach, targeted salivary gland (TSG) immunization, using plasmid pcDNA3/fimA, coding forPorphyromonas gingivalisfimbriae. Expression of subunit protein, fimbrillin, was observed in eukaryotic cells growing in vitro following transfection with pcDNA3/fimA. In this study, we obtained good humoral and cell-mediated immune responses in BALB/c mice by TSG administration using the above-mentioned DNA vaccine. The production of fimbria-specific IgA and IgG antibodies in saliva and serum IgG antibody was significantly stimulated by TSG immunization. Injection of DNA vaccine into salivary gland elicited high-level production of antigen-specific IgG antibody, similar to that induced following intramuscular immunization. The major IgG subclass that recognized fimbriae was IgG2a in serum from pcDNA3/fimA-immunized mice. Reverse transcription-PCR analysis of mononuclear cells from salivary glands showed that levels of Th2 cytokine-specific mRNA were higher in the immunized group than in the nonimmunized group. In addition, TSG DNA immunization resulted in the generation of antigen-specific CTL in spleen. These results indicate that TSG immunization with plasmid DNA may represent a genetic immunization strategy against infection by oral and pharyngeal pathogens that may invade local, mucosal surfaces.
A DNA Sequence Corresponding to the Gene Encoding Cysteine Proteinase 5 in
Entamoeba histolytica
Is Present and Positionally Conserved but Highly Degenerated in
Entamoeba dispar
Authors: Willhoeft Ute , Hamann Lutz , Tannich Egbert 
Published: December 2019

ABSTRACT

Cysteine proteinases of
Entamoeba histolytica
are considered to be one of the most important classes of molecules responsible for the parasite's ability to destroy human tissues. Interestingly, one particular cysteine proteinase, located on the surface of
E. histolytica
trophozoites and designated cysteine proteinase 5 (CP5), is not expressed in the closely related but nonpathogenic species
Entamoeba dispar
. By comparing the
E. histolytica
and
E. dispar
genomic loci containing the gene for CP5 (
cp5
), it was found that the position of
cp5
within the genomic context is conserved between the two organisms, but that the gene is highly degenerated in
E. dispar
, as it contains numerous nucleotide exchanges, insertions, and deletions, resulting in multiple stop codons within the
cp5
reading frame. An alignment of all available orthologous
E. histolytica
and
E. dispar
DNA sequences suggested that
cp5
started to degenerate in
E. dispar
coincidently when the two organisms began to diverge from a common ancestor.
Importance of B cells, but Not Specific Antibodies, in Primary and Secondary Protective Immunity to the Intracellular BacteriumFrancisella tularensisLive Vaccine Strain
Authors: Elkins Karen L. , Bosio Catharine M. , Rhinehart-Jones Tonya R. 
Published: December 2019

ABSTRACTAlthough there appears to be little if any role for specific antibodies in protection against intracellular bacteria, such as the model pathogenF. tularensislive vaccine strain (LVS), the role of B cells themselves in primary and secondary infection with such bacteria has not been examined directly. We show here that mice deficient in mature B cells and antibodies (B-cell knockout mice) are marginally compromised in controlling primary sublethal infection but are 100-fold less well protected against secondary lethal challenge than are their normal counterparts. This defect in optimal specific protective immunity was readily reconstituted by the transfer of primed, and to a lesser degree, unprimed B cells, but not by the transfer of specific antibodies. The results indicate a previously unappreciated role for B cells in secondary immunity to intracellular pathogens through a function other than antibody production.
Laccase ProtectsCryptococcus neoformansfrom Antifungal Activity of Alveolar Macrophages
Authors: Liu Lide , Tewari Ram P. , Williamson Peter R. 
Published: December 2019

ABSTRACTWhile laccase ofCryptococcus neoformansis implicated in the virulence of the organism, our recent studies showing absence of melanin in the infected mouse brain has led us to a search for alternative roles for laccase in cryptococcosis. We investigated the role of laccase in protection ofC. neoformansagainst murine alveolar macrophage (AM)-mediated antifungal activity by using a pair of congenic laccase-positive (2E-TUC) and laccase-deficient (2E-TU) strains. The laccase-positive cells with laccase derepression were more resistant to the antifungal activity of AM than a laccase-deficient strain ([28.9 ± 1.2]% versus [40.2 ± 2.6]% killing). Addition ofl-dopa toCryptococcusto produce melanin in a laccase-positive strain resulted in a slight increase in protection ofC. neoformansfrom the antifungal activity of macrophages ([25.4 ± 3.4]% versus [28.9 ± 1.2]% killing). Recombinant cryptococcal laccase exhibited iron oxidase activity in converting Fe(II) to Fe(III). Moreover, recombinant laccase inhibited killing ofC. neoformansby hydroxyl radicals catalyzed by iron in a cell-free system. Addition of the hydroxyl radical scavenger mannitol or dimethyl sulfoxide to AMs prior to the introduction of cryptococcal cells decreased killing of both strains and reduced the difference in susceptibility between the laccase-positive and laccase-deficient strains. Furthermore, laccase-mediated protection from AM killing was inhibited by the addition of Fe(II), presumably by overcoming the effects of the iron oxidase activity of cryptococcal laccase. These results suggest that the iron oxidase activity of laccase may protectC. neoformansfrom macrophages by oxidation of phagosomal iron to Fe(III) with a resultant decrease in hydroxyl radical formation.