bluuebunny/crossref_metadata_2025_split huggingface dataset:
Shiga Toxin 1 fromEscherichia coliBlocks Activation and Proliferation of Bovine Lymphocyte Subpopulations In Vitro
Authors: Menge C. , Wieler L. H. , Schlapp T. , Baljer G. 
Published: December 2019

ABSTRACTShiga toxin-producingEscherichia coli(STEC) is widespread in the cattle population, but the clinical significance of Shiga toxins (Stx’s) for the bovine species remains obscure. Since Stx’s exert immunomodulating effects in other species, we examined the effect of purified Stx1 on a bovine B lymphoma cell line (BL-3) and peripheral blood mononuclear cells (PBMC) isolated from adult bovine blood by viability assays and flow cytometry analysis. Stx1 markedly induced apoptosis in stimulated BL-3 cells. The susceptibility of this B-cell-derived cell line was induced only by either lipopolysaccharide (LPS) or pokeweed mitogen, while cultures stimulated with T-cell mitogens were unaffected by the toxin. In contrast, Stx1 did not induce cellular death—neither apoptosis nor necrosis—in primary cultures of PBMC but hindered the mitogen-induced increase in metabolic activity. The influence of Stx1 on single PBMC subpopulations varied with the type of mitogenic stimulus applied. Stimulation with phytohemagglutinin P particularly induced the proliferation of bovine CD8-expressing (BoCD8+) cells, and this proliferative response was blocked by Stx1. On the other hand, Stx1 reduced the portion of viable B cells in the presence of LPS. Modulation of activation marker expression (BoCD25 and BoCD71) by Stx1 indicated that the toxin hindered the proliferation of cells by blocking their activation. In conclusion, we assume that Stx1 contributes to the pathogenesis of STEC-associated diarrhea in calves by suppressing the mucosa-associated immune response. The usefulness of cattle as a model in which to study Stx-induced immunomodulation is discussed.
Functional Expression of Nramp1 In Vitro in the Murine Macrophage Line RAW264.7
Authors: Govoni Gregory , Canonne-Hergaux François , Pfeifer Cheryl G. , Marcus Sandra L. , Mills Scott D. , Hackam David J. , Grinstein Sergio , Malo Danielle , Finlay B. Brett , Gros Philippe 
Published: December 2019

ABSTRACTMutations at theNramp1locus in vivo cause susceptibility to infection by unrelated intracellular microbes.Nramp1encodes an integral membrane protein abundantly expressed in the endosomal-lysosomal compartment of macrophages and is recruited to the phagosomal membrane following phagocytosis. The mechanism by which Nramp1 affects the biochemical properties of the phagosome to control microbial replication is unknown. To devise an in vitro assay for Nramp1 function, we introduced a wild-typeNramp1G169cDNA into RAW 264.7 macrophages (which bear a homozygous mutantNramp1D169allele and thus are permissive to replication of specific intracellular parasites). Recombinant Nramp1 was expressed in a membranous compartment in RAW264.7 cells and was recruited to the membrane ofSalmonella typhimuriumandYersinia enterocoliticacontaining phagosomes. Evaluation of the antibacterial activity of RAW264.7 transfectants showed that expression of the recombinant Nramp1 protein abrogated intracellular replication ofS. typhimurium. Studies with a replication-defectiveS. typhimuriummutant suggest that this occurs through an enhanced bacteriostatic activity. The effect of Nramp1 expression was specific, since (i) it was not seen in RAW264.7 transfectants overexpressing the closely related Nramp2 protein, and (ii) control RAW264.7 cells, Nramp1, and Nramp2 transfectants could all efficiently kill a temperature-sensitive, replication-defective mutant ofS. typhimurium. Finally, increased antibacterial activity of the Nramp1 RAW264.7 transfectants was linked to increased phagosomal acidification, a distinguishing feature of primary macrophages expressing a wild-type Nramp1 allele. Together, these results indicate that transfection ofNramp1cDNAs in the RAW264.7 macrophage cell line can be used as a direct assay to study both Nramp1 function and mechanism of action as well as to identify structure-function relationships in this protein.
Replication ofToxoplasma gondii, but NotTrypanosoma cruzi, Is Regulated in Human Fibroblasts Activated with Gamma Interferon: Requirement of a Functional JAK/STAT Pathway
Authors: Cerávolo Isabela Penna , Chaves Andréa C. L. , Bonjardim Cláudio A. , Sibley David , Romanha Alvaro J. , Gazzinelli Ricardo T. 
Published: December 2019

ABSTRACTTo study the role of tryptophan degradation by indoleamine 2,3-dioxygenase (INDO) in the control ofTrypanosoma cruziorToxoplasma gondiireplication, we used human fibroblasts and a fibrosarcoma cell line (2C4). The cells were cultured in the presence or absence of recombinant gamma interferon (rIFN-γ) and/or recombinant tumor necrosis factor alpha (rTNF-α) for 24 h and were then infected with eitherT. cruziorT. gondii. Intracellular parasite replication was evaluated 24 or 48 h after infection. Treatment with rIFN-γ and/or rTNF-α had no inhibitory effect onT. cruzireplication. In contrast, 54, 73, or 30% inhibition ofT. gondiireplication was observed in the cells treated with rIFN-γ alone, rIFN-γ plus rTNF-α, or TNF-α alone, respectively. The replication ofT. gondiitachyzoites in cytokine-activated cells was restored by the addition of extra tryptophan to the culture medium. Similarly,T. gondiitachyzoites transfected with bacterial tryptophan synthase were not sensitive to the microbiostatic effect of rIFN-γ. We also investigated the basis of the cytokine effect on parasite replication by using the three mutant cell lines B3, B9, and B10 derived from 2C4 and expressing defective STAT1α (signal transducer and activator of transcription), JAK2 (Janus family of cytoplasmic tyrosine kinases), or JAK1, respectively, three important elements of a signaling pathway triggered by rIFN-γ. We found that rTNF-α was able to induce low levels expression of INDO mRNA in the parental cell line, as well as the cell line lacking functional JAK2. In contrast to the parental cell line (2C4), rIFN-γ was not able to induce the expression of INDO mRNA or microbiostatic activity in any of the mutant cell lines. These findings indicate the essential requirement of the JAK/STAT pathway for the induction of high levels of INDO mRNA, tryptophan degradation, and the anti-Toxoplasmaactivity inside human nonprofessional phagocytic cells.
Altered Immune Response of Interferon Regulatory Factor 1-Deficient Mice againstPlasmodium bergheiBlood-Stage Malaria Infection
Authors: Tan Rosemary Sok-Pin , Feng Chiguang , Asano Yoshihiro , Kara Anna Ursula 
Published: December 2019

ABSTRACTNitric oxide (NO) is a short-lived biological mediator which can be induced in various cell types and is able to cause many metabolic changes in target cells. Inhibition of tumor cell growth and antimicrobial activity has been attributed to the stimulation of NO production by transcriptional upregulation of inducible nitric oxide synthase. In the present study, we used mice devoid of functional interferon regulatory factor 1 by targeted gene disruption (IRF-1−/−) to investigate the role of NO in the host immune response against blood-stagePlasmodium bergheiANKA infection. IRF-1−/−mice survived longer with a later onset of and a lower peak parasitemia despite the inability to produce appreciable levels of NO. The administration of exogenous interleukin-12 (IL-12) was able to prolong survival in the wild-type mice with an upregulation in the expression of both gamma interferon (IFN-γ) and NO. However, the administration of IL-12 did not improve the survival of IRF-1−/−mice. These studies indicate that while IL-12 is able to mediate protection via an IFN-γ- and NO-dependent pathway in the wild-type mice, such a protective mechanism may not be functional in the IRF-1−/−mice. Our results suggest that NO may not be essential for host immunity to the parasite and that IRF-1−/−mice are able to induce an IFN-γ- and NO-independent mechanism againstP. bergheiinfection.
Relationship between Cell Surface Carbohydrates and Intrastrain Variation on Opsonophagocytosis ofStreptococcus pneumoniae
Authors: Kim Jean O. , Romero-Steiner Sandra , Sørensen Uffe B. Skov , Blom Jens , Carvalho M. , Barnard S. , Carlone George , Weiser Jeffrey N. 
Published: December 2019

ABSTRACTStreptococcus pneumoniaeundergoes spontaneous phase variation between a transparent and an opaque colony phenotype, the latter being more virulent in a murine model of sepsis. Opaque pneumococci have previously been shown to express lower amounts of C polysaccharide (cell wall teichoic acid) and in this study were shown to have a higher content of capsular polysaccharide by immunoelectron microscopy. This report then examined the relationship between expression of these two cell surface carbohydrate structures and their relative contribution to the increased virulence of opaque variants. Comparison of genetically related strains showed that the differential content of capsular polysaccharide did not affect the amount of teichoic acid as measured by a capture enzyme-linked immunosorbent assay (ELISA). In contrast, when the teichoic acid structure was altered by replacing choline in the growth medium with structural analogs, the quantity of capsular polysaccharide as measured by a capture ELISA was decreased, demonstrating a linkage in the expression of the two surface carbohydrate structures. A standardized assay was used to assess the relative contribution of cell surface carbohydrates to opsonophagocytosis. The opaque variants required 1.2- to 30-fold more immune human serum to achieve 50% opsonophagocytic killing than did related transparent variants (types 6B and 9V). The opsonophagocytic titer was proportional to the quantity of capsular polysaccharide rather than teichoic acid. The major factor in binding of the opsonin, C-reactive protein (CRP), was also the amount of capsular polysaccharide rather than the teichoic acid ligand. Only for the transparent variant (type 6B), which bound more CRP, was there enhanced opsonophagocytic killing in the presence of this serum protein. Increased expression of capsular polysaccharide, therefore, appeared to be the major factor in the decreased opsonophagocytic killing of opaque pneumococci.
Zinc-Regulated Biosynthesis of Immunodominant Antigens from
Aspergillus
spp
Authors: Segurado Mónica , López-Aragón Raquel , Calera José Antonio , Fernández-Abalos José Manuel , Leal Fernando 
Published: December 2019

ABSTRACT

ASPND1 and ASPF2 are immunodominant antigens from
Aspergillus nidulans
and
A. fumigatus
, respectively, that are readily synthesized in infections in the human host, as demonstrated by their reactivity with more than 80% of sera from patients with aspergilloma or allergic bronchopulmonary aspergillosis. We demonstrate here that both antigens are exclusively produced under situations of low bioavailability of free Zn
2+
. Addition of micromolar concentrations of Zn
2+
to the culture medium strongly stimulated
Aspergillus
growth but totally inhibited ASPND1 or ASPF2 production. This effect was specific, since other divalent metals had no effect. Removal of endogenous Zn
2+
by a chelator also stimulated ASPND1 production, and the effect was specifically reversed by Zn
2+
. These results suggest a possible role of these antigens in the survival of the fungus in the lungs.
Enterohemorrhagic
Escherichia coli
O157:H7 Produces Tir, Which Is Translocated to the Host Cell Membrane but Is Not Tyrosine Phosphorylated
Authors: DeVinney Rebekah , Stein Markus , Reinscheid Dieter , Abe Akio , Ruschkowski Sharon , Finlay B. Brett 
Published: December 2019

ABSTRACT

Intimate attachment to the host cell leading to the formation of attaching and effacing (A/E) lesions is an essential feature of enterohemorrhagic
Escherichia coli
(EHEC) O157:H7 pathogenesis. In a related pathogen, enteropathogenic
E. coli
(EPEC), this activity is dependent upon translocation of the intimin receptor, Tir, which becomes tyrosine phosphorylated within the host cell membrane. In contrast, the accumulation of tyrosine-phosphorylated proteins beneath adherent EHEC bacteria does not occur, leading to questions about whether EHEC uses a Tir-based mechanism for adherence and A/E lesion formation. In this report, we demonstrate that EHEC produces a functional Tir that is inserted into host cell membranes, where it serves as an intimin receptor. However, unlike in EPEC, in EHEC Tir is not tyrosine phosphorylated yet plays a key role in both bacterial adherence to epithelial cells and pedestal formation. EHEC, but not EPEC, was unable to synthesize Tir in Luria-Bertani medium but was able to secrete Tir into M9 medium, suggesting that Tir synthesis and secretion may be regulated differently in these two pathogens. EHEC Tir and EPEC Tir both bind intimin and focus cytoskeletal rearrangements, indicating that tyrosine phosphorylation is not needed for pedestal formation. EHEC and EPEC intimins are functionally interchangeable, but EHEC Tir shows a much greater affinity for EHEC intimin than for EPEC intimin. These findings highlight some of the differences and similarities between EHEC and EPEC virulence mechanisms, which can be exploited to further define the molecular basis of pedestal formation.
Immune Response toNocardia brasiliensisAntigens in an Experimental Model of Actinomycetoma in BALB/c Mice
Authors: Salinas-Carmona Mario C. , Torres-Lopez Ernesto , Ramos Alma I. , Licon-Trillo Angel , Gonzalez-Spencer Daniel 
Published: December 2019

ABSTRACTNine- to twelve-week-old BALB/c mice were injected in footpads with 107CFU of aNocardia brasiliensiscell suspension. Typical actinomycetoma lesions, characterized by severe local inflammation with abscess and fistula formation, were fully established by day 28 after infection. These changes presented for 90 days, and then tissue repair with scar formation slowly appeared, with complete healing after 150 days of infection. Some animals developed bone destruction in the affected area. Histopathology showed an intense inflammatory response, with polymorphonuclear cells and hyaloid material around the colonies of the bacteria, some of which were discharged from draining abscesses. Sera from experimental animals were analyzed by Western blotting, and immunodominant antigens P61 and P24 were found as major targets for antibody response. Anti-P24 immunoglobulin M (IgM) isotype antibodies were present as early as 7 days, IgG peaking 45 days after infection. Lymphocyte proliferation with spleen and popliteal lymph node cells demonstrated thymidine incorporation at 7 days after infection, the stimulation index decreasing by day 60. Levels of interleukin-1 (IL-1), IL-2, IL-4, IL-6, tumor necrosis factor alpha, and gamma interferon (IFN-γ) were determined by enzyme-linked immunosorbent assay in the sera of infected animals. The circulating levels of IFN-γ increased more than 10 times the basal levels; levels of IL-4, IL-6 and IL-10 also increased during the first 4 days of infection.
Insertional Inactivation of Genes Responsible for the
d
-Alanylation of Lipoteichoic Acid in
Streptococcus gordonii
DL1 (Challis) Affects Intrageneric Coaggregations
Authors: Clemans Daniel L. , Kolenbrander Paul E. , Debabov Dmitri V. , Zhang Qunying , Lunsford R. Dwayne , Sakone Holly , Whittaker Catherine J. , Heaton Michael P. , Neuhaus Francis C. 
Published: December 2019

ABSTRACT

Most human oral viridans streptococci participate in intrageneric coaggregations, the cell-to-cell adherence among genetically distinct streptococci. Two genes relevant to these intrageneric coaggregations were identified by transposon Tn
916
mutagenesis of
Streptococcus gordonii
DL1 (Challis). A 626-bp sequence flanking the left end of the transposon was homologous to
dltA
and
dltB
of
Lactobacillus rhamnosus
ATCC 7469 (formerly called
Lactobacillus casei
). A 60-kb probe based on this flanking sequence was used to identify the homologous DNA in a fosmid library of
S. gordonii
DL1. This DNA encoded
d
-alanine-
d
-alanyl carrier protein ligase that was expressed in
Escherichia coli
from the fosmid clone. The cloned streptococcal
dltA
was disrupted by inserting an
ermAM
cassette, and then it was linearized and transformed into
S. gordonii
DL1 for allelic replacement. Erythromycin-resistant transformants containing a single insertion in
dltA
exhibited a loss of
d
-alanyl esters in lipoteichoic acid (LTA) and a loss of intrageneric coaggregation. This phenotype was correlated with the loss of a 100-kDa surface protein reported previously to be involved in mediating intrageneric coaggregation (C. J. Whittaker, D. L. Clemans, and P. E. Kolenbrander, Infect. Immun. 64:4137–4142, 1996). The mutants retained the parental ability to participate in intergeneric coaggregation with human oral actinomyces, indicating the specificity of the mutation in altering intrageneric coaggregations. The mutants were altered morphologically and exhibited aberrant cell septa in a variety of pleomorphs. The natural DNA transformation frequency was reduced 10-fold in these mutants. Southern analysis of chromosomal DNAs from various streptococcal species with the
dltA
probe revealed the presence of this gene in most viridans streptococci. Thus, it is hypothesized that
d
-alanyl LTA may provide binding sites for the putative 100-kDa adhesin and scaffolding for the proper presentation of this adhesin to mediate intrageneric coaggregation.
Expression of Interleukin-6 in the Cornea in Response to Infection with Different Strains ofPseudomonas aeruginosa
Authors: Cole Nerida , Bao Shisan , Willcox Mark , Husband Alan J. 
Published: December 2019

ABSTRACTStrains ofPseudomonas aeruginosacausing keratitis can be either cytotoxic (6206) or invasive (6294), while a strain (Paer1) causing contact lens-induced acute red eye has been shown to be neither. In situ hybridization was used to examine the location and identity of cells expressing interleukin-6 (IL-6) mRNA in the murine cornea and changes in expression in response to infection with different strains ofP. aeruginosa. The number of IL-6-positive cells was determined by image analysis. IL-6 protein levels were measured by an enzyme-linked immunosorbent assay. BALB/c mice were challenged by use of the wounded-cornea model withP. aeruginosa6294, 6206, or Paer1 (2 × 106CFU). At time intervals up to 24 h, postchallenge corneal tissue was probed for IL-6 mRNA. IL-6 mRNA expression was rapidly elevated in the epithelium in response to strains 6294 and 6206. At the conclusion of the experiments, infiltrating inflammatory cells also stained positively for IL-6 mRNA. In contrast, corneas challenged with strain Paer1 showed significant upregulation of IL-6 mRNA only at 4 h postchallenge. Three distinct patterns of IL-6 mRNA expression in the mouse cornea occur in response to these three ocular isolates ofP. aeruginosa. The data obtained for mRNA expression in the cornea for all three strains ofP. aeruginosacorrelated well with IL-6 protein analysis of whole-eye homogenates. Differences in the cytokine responses to these strains correlate with differences in the pathology associated with each strain and may offer an opportunity to develop strategies for the improved management of ocular inflammation.
Local Secretory Immunoglobulin A and Postimmunization Gastritis Correlate with Protection againstHelicobacter pyloriInfection after Oral Vaccination of Mice
Authors: Goto Takayuki , Nishizono Akira , Fujioka Toshio , Ikewaki Junko , Mifune Kumato , Nasu Masaru 
Published: December 2019

ABSTRACTC57BL/6 mice were orally immunized with five weekly doses of 2 mg, 200 μg, or 2 μg ofHelicobacter pylori(Sydney strain) whole-cell sonicate combined with cholera toxin. One week after the last vaccination, mice were challenged with 5 × 107CFU of liveH. pylorithree times at 2-day intervals. At 6 or 18 weeks after the challenge, mice were sacrificed and bacterial cultures and histological studies of the stomach were performed. Vaccination with 2 mg/session or 200 μg/session inhibitedH. pyloricolonization by 90 and 100%, respectively. These mice were considered protected. Lower levels ofH. pylori-specific immunoglobulin A (IgA) were detected in fecal and saliva samples before challenge. However, a significant increase in IgA secretion in mucosal tissue and a higher labeling index for IgA-positive lumina of pyloric glands were noted in these mice in response to challenge and in a vaccine dose-dependent manner. In protected mice, however, severe gastritis characterized by marked infiltration of inflammation mononuclear cells was noted at 6 weeks after challenge, compared with the gastritis seen in unprotected mice or nonvaccinated, ordinarily infected mice. Marked expression of gamma interferon mRNA was detected in the stomach of all protected mice, and 50% of these mice expressed interleukin 4 (IL-4) or IL-5 mRNA. Our findings suggest that local secretory IgA antibody and severe postimmunization gastritis correlate well with protection of mice againstH. pyloriinfection.
Lipopolysaccharide Induction of Tissue Factor Expression in Rabbits
Authors: Erlich Jonathan , Fearns Colleen , Mathison John , Ulevitch Richard J. , Mackman Nigel 
Published: December 2019

ABSTRACT
Tissue factor (TF) is the major activator of the coagulation protease cascade and contributes to lethality in sepsis. Despite several studies analyzing TF expression in animal models of endotoxemia, there remains debate about the cell types that are induced to express TF in different tissues. In this study, we performed a detailed analysis of the induction of TF mRNA and protein expression in two rabbit models of endotoxemia to better understand the cell types that may contribute to local fibrin deposition and disseminated intravascular coagulation. Northern blot analysis demonstrated that lipopolysaccharide (LPS) increased TF expression in the brain, lung, and kidney. In situ hybridization showed that TF mRNA expression was increased in cells identified morphologically as epithelial cells in the lung and as astrocytes in the brain. In the kidney, in situ hybridization experiments and immunohistochemical analysis showed that TF mRNA and protein expression was increased in renal glomeruli and induced in tubular epithelium. Dual staining for TF and vWF failed to demonstrate TF expression in endothelial cells in LPS-treated animals. These results demonstrate that TF expression is induced in many different cell types in LPS-treated rabbits, which may contribute to local fibrin deposition and tissue injury during endotoxemia.
Role of Interleukin-18 (IL-18) in Mycobacterial Infection in IL-18-Gene-Disrupted Mice
Authors: Sugawara Isamu , Yamada Hiroyuki , Kaneko Hirotaka , Mizuno Satoru , Takeda Kiyoshi , Akira Shizuo 
Published: December 2019

ABSTRACTImmunity to mycobacterial infection is closely linked to the emergence of T cells that secrete cytokines, gamma interferon (IFN-γ), interleukin-12 (IL-12), and tumor necrosis factor alpha (TNF-α), resulting in macrophage activation and recruitment of circulating monocytes to initiate chronic granuloma formation. The cytokine that mediates macrophage activation is IFN-γ, and, like IL-12, IL-18 was shown to activate Th1 cells and induce IFN-γ production by these cells. In order to investigate the role of IL-18 in mycobacterial infection, IL-18-deficient mice were infected withMycobacterium tuberculosisandMycobacterium bovisBCG Pasteur, and their capacities to control bacterial growth, granuloma formation, cytokine secretion, and NO production were examined. These mice developed marked granulomatous, but not necrotic, lesions in their lungs and spleens. Compared with the levels in wild-type mice, the splenic IFN-γ levels were low but the IL-12 levels were normal in IL-18-deficient mice. The reduced IFN-γ production was not secondary to reduced induction of IL-12 production. The levels of NO production by peritoneal macrophages of IL-18-deficient and wild-type mice did not differ significantly. Granulomatous lesion development by IL-18-deficient mice was inhibited significantly by treatment with exogenous recombinant IL-18. Therefore, IL-18 is important for the generation of protective immunity to mycobacteria, and its main function is the induction of IFN-γ expression.