bluuebunny/crossref_metadata_2025 huggingface dataset:
Pseudomonas aeruginosa
Quorum-Sensing Signal Molecule
N
-(3-Oxododecanoyl)-
l
-Homoserine Lactone Inhibits Expression of P2Y Receptors in Cystic Fibrosis Tracheal Gland Cells
Authors: Saleh A. , Figarella C. , Kammouni W. , Marchand-Pinatel S. , Lazdunski A. , Tubul A. , Brun P. , Merten M. D.
Published: December 2019

ATP and UTP have been proposed for use as therapeutic treatment of the abnormal ion transport in the airway epithelium in cystic fibrosis (CF), the most characteristic feature of which is permanent infection by
Pseudomonas aeruginosa
. As for diverse gram-negative bacteria, this pathogenic bacterium accumulates diffusible
N
-acylhomoserine lactone (AHL) signal molecules, and when a threshold concentration is reached, virulence factor genes are activated. Human submucosal tracheal gland serous (HTGS) cells are believed to play a major role in the physiopathology of CF. Since ATP and UTP stimulate CF epithelial cells through P2Y receptors, we sought to determine whether CF HTGS cells are capable of responding to the AHLs
N
-butanoyl-
l
-homoserine lactone (BHL),
N
-hexanoyl-
l
-homoserine lactone (HHL),
N
-(3-oxododecanoyl)-
l
-homoserine lactone (OdDHL), and
N
-(3-oxohexanoyl)-
l
-homoserine lactone (OHHL), with special reference to P2Y receptors. All AHLs inhibited ATP- and UTP-induced secretion by CF HTGS cells. The 50% inhibitory concentrations were as high as 10 and 5 μM for BHL and HHL, respectively, but were only 0.3 and 0.4 pM for OdDHL and OHHL, respectively. Furthermore, all AHLs down-regulated the expression of the P2Y2 and P2Y4 receptors. Ibuprofen and nordihydroguaiaretic acid were able to prevent AHL inhibition of the responses to nucleotides, but neither dexamethasone nor indomethacin was able to do this. These data indicate that AHLs may alter responsiveness to ATP and UTP by CF HTGS cells and suggest that, in addition to ATP and/or UTP analogues, ibuprofen may be of use for a combinational pharmacological therapy for CF.
Kinetics and Mechanisms of Extracellular Protein Release by
Helicobacter pylori
Authors: Schraw Wayne , McClain Mark S. , Cover Timothy L.
Published: December 2019

To investigate the kinetics and mechanisms of extracellular protein release by
Helicobacter pylori
, we analyzed the entry of metabolically radiolabeled bacterial proteins into broth culture supernatant. At early time points, vacuolating cytotoxin (VacA) constituted a major extracellular protein. Subsequently, culture supernatants accumulated many proteins that were components of intact bacterial cells. This nonselective release of proteins was associated with a decreasing turbidity of cultures and loss of bacterial viability, indicative of an autolytic process. The rates of VacA secretion and autolysis were each influenced by medium composition, and therefore these may be regulated phenomena. Extracellular release of proteins by
H. pylori
may be an important adaptation that facilitates the persistence of
H. pylori
in the human gastric mucus layer. Moreover, entry of proinflammatory proteins into the gastric mucosa may contribute to the induction of a mucosal inflammatory response.
Role ofLeishmania donovaniand Its Lipophosphoglycan in CD4+T-cell Activation-Induced Human Immunodeficiency Virus Replication
Authors: Wolday Dawit , Akuffo Hannah , Demissie Abebech , Britton Sven
Published: December 2019

Chronic immune activation by coinfecting pathogens has been suggested as a cofactor in human immunodeficiency virus (HIV) disease progression, particularly in the setting of developing countries. Here, we used in vivo-infected mononuclear cells to examine the role of the protozoan parasiteLeishmania donovaniand its major membrane constituent, lipophosphoglycan (LPG), in mediating CD4+T-lymphocyte activation-induced HIV replication and CD4+T-cell death. We found thatLeishmaniaantigens upregulated HIV replication in CD8-depleted peripheral blood mononuclear cells from asymptomatic HIV-infected donors compared to unstimulated cells.L. donovani-induced viral replication was associated with cellular proliferation, increased expression of the cellular immune activation markers CD25 and HLA-DR within the CD4+subpopulation, and enhanced secretion of tumor necrosis factor alpha (TNF-α), interleukin 2 (IL-2), and IL-6. LPG induced TNF-α secretion in the absence of increased expression of cellular activation markers. Moreover, in a few cases we observed thatL. donovaniinduced HIV replication without significant cellular activation but with cytokine secretion. The rate of apoptosis was accelerated in these latently infected CD4+T cells primed withLeishmaniaantigens compared to controls, and TNF-α production appeared to be the central event necessary for this effect. Furthermore, we demonstrate that thalidomide inhibitedLeishmania-induced virus replication coupled with abrogatedLeishmania-induced TNF-α secretion but not IL-2 or IL-6 production. Furthermore, thalidomide did not affectLeishmania-induced apoptosis. The results suggest thatLeishmaniaand its product, LPG, up-regulate HIV replication in latently infected cells through distinct antigen-specific and non-antigen-specific cellular immune activation mechanisms and that TNF-α secretion is pivotal in this process. The immunomodulatory role of thalidomide raises interest as a potential adjuvant to reduce HIV disease progression inLeishmania-HIV coinfected individuals.
Trapping and Immobilization ofNippostrongylus brasiliensisLarvae at the Site of Inoculation in Primary Infections of Interleukin-5 Transgenic Mice
Authors: Daly Christine M. , Mayrhofer Graham , Dent Lindsay A.
Published: December 2019

Interleukin-5 (IL-5) transgenic mice are highly resistant to primary infections with the intestinal nematodeNippostrongylus brasiliensis; few parasites are found in the intestines of infected animals, and egg production is minimal. While adult worms may be damaged in the intestine, larval migration, development, and viability may also be impaired in other tissues. This study addresses the migration ofN. brasiliensislarvae through the skin and lungs and associated cellular responses in primary infections of IL-5 transgenic mice. Although some larvae may have been trapped and killed in the lungs of IL-5 transgenic mice, most apparently failed to reach this site. Two or more hours after infection of IL-5 transgenic mice, eosinophils were a major component of the cellular infiltrate at the subcutaneous site of injection, and localized eosinophil degranulation was extensive. Seventy-five to ninety-five percent of the larvae injected into subcutaneous air pouches in IL-5 transgenic mice were retained there for at least 24 h. In contrast, in nontransgenic mice, less than 20% of larvae could be recovered from the skin 2 or more h postinjection, and eosinophil activity was modest at all times. The data strongly suggest that eosinophils can restrict the movement ofN. brasiliensislarvae in the first few hours of a primary infection and that this has profound effects on later stages of parasite development. Preexisting eosinophilia, due either to allergy or to infection with tissue-invasive helminth species, may therefore confer some degree of immediate and nonspecific resistance in primary infections with parasitic worms.
Fasciola hepaticaSuppresses a Protective Th1 Response againstBordetella pertussis
Authors: Brady Miriam T. , O’Neill Sandra M. , Dalton John P. , Mills Kingston H. G.
Published: December 2019

Fasciolosis, like other helminth infections, is associated with the induction of T-cell responses polarized to the Th2 subtype. Respiratory infection withBordetella pertussisor immunization with a pertussis whole-cell vaccine (Pw) induces a potent Th1 response, which confers a high level of protection against bacterial challenge. We have used these two pathogens to examine bystander cross-regulation of Th1 and Th2 cells in vivo and provide evidence of immunomodulation of host T-cell responses toB. pertussisby a concomitant infection withFasciola hepatica. Mice with a coinfection ofF. hepaticaandB. pertussisexhibited a Th2 cytokine profile in response toF. hepaticaantigens, similar to those infected withF. hepaticaalone. By contrast, the Th1 response toB. pertussisantigens was markedly suppressed and the bacterial infection was exacerbated following infection withF. hepatica. Furthermore, an established Th1 response induced in mice by infection withB. pertussisor by parenteral immunization with Pw was also suppressed following infection withF. hepatica. This immunomodulatory effect ofB. pertussis-induced responses byF. hepaticainfection is significantly reduced, but not completely abrogated, in IL-4 knockout mice. Our findings demonstrate that Th2-inducing parasites can exert bystander suppression of protective Th1 responses to infection or vaccination with a bacterial pathogen and that the modulation is mediated in part by IL-4 and, significantly, is effective at both the induction and effector stages of the Th1 response.
Significance of Host Cell Kinesin in the Development of
Chlamydia psittaci
Authors: Escalante-Ochoa Cristina , Ducatelle Richard , Charlier Gerard , De Vos Kurt , Haesebrouck Freddy
Published: December 2019

The influence of the microtubule-associated motor protein kinesin on
Chlamydia psittaci
inclusion development in epithelial and fibroblast cell lines was addressed. Kinesin was blocked early after chlamydial internalization (4 h postinfection [p.i.]) and before the initiation of active chlamydial multiplication (8 h p.i.). Chlamydia development was monitored by fluorescence and transmission electron microscopy at different times during the cycle. In both host cell lines, kinesin blockage restricted mitochondria from the chlamydial vacuole. The effects of kinesin blockage on the
C. psittaci
replication cycle included the presence of multiple inclusions up to late in the cycle, the presence of enlarged pleomorphic reticulate bodies, and a delayed reappearance of elementary bodies. The last effect seems to be greater when kinesin is blocked early after infection. Our results show that kinesin activity is required for optimal development of these microorganisms, most probably acting through the apposition of mitochondria to the
C. psittaci
inclusions.
Escherichia coli
Binding to and Invasion of Brain Microvascular Endothelial Cells Derived from Humans and Rats of Different Ages
Authors: Stins Monique F. , Nemani Prasadarao V. , Wass Carol , Kim Kwang Sik
Published: December 2019

Escherichia coli
meningitis commonly occurs in the neonatal period, but the basis of this age dependency is unclear. We have previously identified two types of
E. coli
-brain microvascular endothelial cell (BMEC) interactions contributing to
E. coli
traversal of the blood-brain barrier (i.e., binding and invasion). The present study examined whether the age dependency of
E. coli
meningitis stemmed from differences in the capacities of neonatal and adult BMECs to interact with
E. coli
. BMECs were isolated from rats of different ages (10 days, 20 days and 3 months) as well as from humans of different ages (fetuses, 4- to 7-year-old children, and a 35-year-old adult, and 60- to 85-year-old geriatrics). The bindings of
E. coli
to young and old rat BMECs were similar. Also, the abilities of
E. coli
to invade BMECs were similar for BMECs derived from young and old rats and from human fetuses, children, adults, and geriatrics. These findings suggest that the predominance of
E. coli
meningitis in neonates is not likely due to greater binding and invasion capacities of newborn compared to adult BMECs.
Carrier Properties of a Protein Derived from Outer Membrane Protein A ofKlebsiella pneumoniae
Authors: Rauly Isabelle , Goetsch Liliane , Haeuw Jean-François , Tardieux Christine , Baussant Thierry , Bonnefoy Jean-Yves , Corvaia Nathalie
Published: December 2019

We have recently cloned a new protein, recombinant P40 (rP40). When tested in vivo after conjugation to a B-cell epitope, rP40 induces an important antibody response without the need for adjuvant. To characterize its potency, this carrier protein was coupled to a peptide derived from respiratory syncytial virus attachment G protein (G1′). After immunization of mice with the rP40-G1′ conjugate, strong antipeptide antibodies were detected, whereas peptide alone was not immunogenic. To emphasize the carrier properties of rP40, a polysaccharide derived fromHaemophilus influenzaetype b (Hib) was coupled to it. Immunoglobulin G responses against the Hib polysaccharide were observed after coupling to rP40. Interestingly, an antipeptide antibody response was observed despite preexisting anti-rP40 antibodies generated by preimmunization with rP40. In addition, rP40 compares well with the reference carrier protein, tetanus toxoid (TT), since antibody responses of equal intensity were observed when a peptide or a polysaccharide was coupled to TT and rP40. Moreover, rP40 had advantages compared to TT; e.g., it induced a mixed Th1/Th2 response, whereas TT induced only a Th2 profile. Together, the results indicate that rP40 is a novel carrier protein with potential for use as an alternative carrier for human vaccination.
Identification by Subtractive Hybridization of a Novel Insertion Sequence Specific for Virulent Strains of
Porphyromonas gingivalis
Authors: Sawada Koichi , Kokeguchi Susumu , Hongyo Hiroshi , Sawada Satoko , Miyamoto Manabu , Maeda Hiroshi , Nishimura Fusanori , Takashiba Shogo , Murayama Yoji
Published: December 2019

Subtractive hybridization was employed to isolate specific genes from virulent
Porphyromonas gingivalis
strains that are possibly related to abscess formation. The genomic DNA from the virulent strain
P. gingivalis
W83 was subtracted with DNA from the avirulent strain ATCC 33277. Three clones unique to strain W83 were isolated and sequenced. The cloned DNA fragments were 885, 369, and 132 bp and had slight homology with only
Bacillus stearothermophilus
IS
5377
, which is a putative transposase. The regions flanking the cloned DNA fragments were isolated and sequenced, and the gene structure around the clones was revealed. These three clones were located side-by-side in a gene reported as an outer membrane protein. The three clones interrupt the open reading frame of the outer membrane protein gene. This inserted DNA, consisting of three isolated clones, was designated IS
1598
, which was 1,396 bp (i.e., a 1,158-bp open reading frame) in length and was flanked by 16-bp terminal inverted repeats and a 9-bp duplicated target sequence. IS
1598
was detected in
P. gingivalis
W83, W50, and FDC 381 by Southern hybridization. All three
P. gingivalis
strains have been shown to possess abscess-forming ability in animal models. However, IS
1598
was not detected in avirulent strains of
P. gingivalis
, including ATCC 33277. The IS
1598
may interrupt the synthesis of the outer membrane protein, resulting in changes in the structure of the bacterial outer membrane. The IS
1598
isolated in this study is a novel insertion element which might be a specific marker for virulent
P. gingivalis
strains.
Analysis of Virulence of Clinical Isolates ofSalmonella enteritidisIn Vivo and In Vitro
Authors: Lu Sangwei , Manges Amee R. , Xu Yisheng , Fang Ferric C. , Riley Lee W.
Published: December 2019

Salmonella entericaserotype Enteritidis (S. enteritidis) is a major food-borne pathogen, and its incidence among allSalmonellaserotypes has increased dramatically in the last two decades. To study the virulence characteristics of clinical isolates ofS. enteritidis, we determined the 50% lethal doses (LD50) in mice of isolates of two major phage types (4 and 8). Isolates of both phage types showed a wide range of LD50after oral inoculation, varying from under 102organisms to over 108organisms. No significant difference in LD50was observed between the phage types. These observations indicated that clinical isolates ofS. enteritidisare highly heterogeneous in their ability to cause death in mice. We compared the LD50s of these isolates to the results observed from in vitro pathogenicity assays. We also analyzed these isolates for recognizedSalmonellavirulence loci (spv,sodCI,sopE, andsef). The in vitro phenotypes of the isolates showed no obvious correlation with their LD50in any given assay, and the virulence genes tested were present in all isolates. However, the isolate with the lowest LD50(isolate 97A 2472) was resistant to acidified sodium nitrite (ASN). Moreover, the most acid-susceptible, macrophage-susceptible, and ASN-susceptible isolates were attenuated for virulence in mice. These results, based on extensive analysis of clinical isolates ofS. enteritidis, demonstrate the complex nature ofSalmonellapathogenesis in mice. Our results also indicate the limitation of in vitro assays in predicting in vivo virulence.
Differences in Surface Expression of NspA amongNeisseria meningitidisGroup B Strains
Authors: Moe Gregory R. , Tan Siqi , Granoff Dan M.
Published: December 2019

NspA is a highly conserved membrane protein that is reported to elicit protective antibody responses againstNeisseria meningitidisserogroups A, B and C in mice (D. Martin, N. Cadieux, J. Hanel, and B. R. Brodeur, J. Exp. Med. 185:1173–1183, 1997). To investigate the vaccine potential of NspA, we produced mouse anti-recombinant NspA (rNspA) antisera, which were used to evaluate the accessibility of NspA epitopes on the surface of different serogroup B strains by an immunofluorescence flow cytometric assay and by susceptibility to antibody-dependent, complement-mediated bacteriolysis. Among 17 genetically diverse strains tested, 11 (65%) were positive for NspA cell surface epitopes and 6 (35%) were negative. All six negative strains also were resistant to bactericidal activity induced by the anti-rNspA antiserum. In contrast, of the 11 NspA surface-positive strains, 8 (73%;P< 0.05) were killed by the antiserum and complement. In infant rats challenged with one of these eight strains, the anti-rNspA antiserum conferred protection against bacteremia, whereas the antiserum failed to protect rats challenged by one of the six NspA cell surface-negative strains. Neither NspA expression nor protein sequence accounted for differences in NspA surface accessibility, since all six negative strains expressed NspA in outer membrane preparations and since their predicted NspA amino acid sequences were 99 to 100% identical to those of three representative positive strains. However, the six NspA cell surface-negative strains produced, on average, larger amounts of group B polysaccharide than did the 11 positive strains (reciprocal geometric mean titers, 676 and 224, respectively;P< 0.05), which suggests that the capsule may limit the accessibility of NspA surface epitopes. Given these strain differences in NspA surface accessibility, an rNspA-based meningococcal B vaccine may have to be supplemented by additional antigens.
Interaction ofMycobacterium tuberculosis-Induced Transforming Growth Factor β1 and Interleukin-10
Authors: Othieno Catherine , Hirsch Christina S. , Hamilton Beverly D. , Wilkinson Katalin , Ellner Jerrold J. , Toossi Zahra
Published: December 2019

Mycobacterium tuberculosisis associated with the activation of cytokine circuits both at sites of active tuberculosis in vivo and in cultures of mononuclear cells stimulated byM. tuberculosisor its components in vitro. Interactive stimulatory and/or inhibitory pathways are established between cytokines, which may result in potentiation or attenuation of the effects of each molecule on T-cell responses. Here we examined the interaction of transforming growth factor β1 (TGF-β1) and interleukin-10 (IL-10) in purified protein derivative (PPD)-stimulated human mononuclear cell cultures in vitro. TGF-β1 induced monocyte IL-10 (but not tumor necrosis factor alpha) production (by 70-fold,P< 0.02) and mRNA expression in the absence but not in the presence of PPD. Both exogenous recombinant (r) IL-10 and rTGF-β1 independently suppressed the production of PPD-induced gamma interferon (IFN-γ) in mononuclear cells from PPD skin test-positive individuals. Synergistic suppression of IFN-γ in cultures containing both rTGF-β1 and rIL-10 was only seen when the responder cell population were peripheral blood mononuclear cells (PBMC) and not monocyte-depleted mononuclear cells and when PBMC were pretreated with rTGF-β1 but not with rIL-10. Suppression of PPD-induced IFN-γ in PBMC containing both rTGF-β1 (1 ng/ml) and rIL-10 (100 pg/ml) was 1.5-fold higher (P< 0.05) than cultures containing TGF-β1 alone and 5.7-fold higher (P< 0.004) than cultures containing IL-10 alone. Also, neutralization of endogenous TGF-β1 and IL-10 together enhanced PPD-induced IFN-γ in PBMC in a synergistic manner. Thus, TGF-β1 and IL-10 together potentiate the downmodulatory effect onM. tuberculosis-induced T-cell production of IFN-γ, and TGF-β1 alone enhances IL-10 production. At sites of activeM. tuberculosisinfection, these interactions may be conducive to the suppression of mononuclear cell functions.
Upregulation of p75 Tumor Necrosis Factor Alpha Receptor inMycobacterium avium-Infected Mice: Evidence for a Functional Role
Authors: Corti Angelo , Fattorini Lanfranco , Thoresen Ove Fredrik , Ricci Maria Luisa , Gallizia Anna , Pelagi Micaela , Li Yongjun , Orefici Graziella
Published: December 2019

The bacterial growth and the production of tumor necrosis factor alpha (TNF-α) and TNF receptors (TNF-Rs) in the spleen and blood of BALB/c mice challenged withMycobacterium aviumcomplex (MAC) were monitored. Infection developed in two phases: the first, up to day 21, was associated with rapid MAC multiplication in the spleen and a drop in the mycobacteremia, and the second was associated with control of the infection in both compartments. In the spleen, TNF-α and TNF-RII mRNA levels peaked on day 21 and then slowly decreased; however, no increase in the level of TNF-RI mRNA was observed throughout these experiments. The level of circulating soluble TNF-RII (sTNF-RII) was transiently increased after day 21. In a model in which overproduction of bioactive TNF-α was triggered in response to a second infection with MAC, an increased production of sTNF-RII by cultured splenocytes was also observed. Administration of an antagonist anti-TNF-RII monoclonal antibody (MAb 6G1) to infected mice inhibited the bacterial growth in the spleen, suggesting that the TNF-RII and/or sTNF-RII was functionally involved in the mechanisms that control the infection. Overall, these observations suggest that upregulation of TNF-RII or sTNF-RII contributes to modulation of the TNF-α antibacterial activity in MAC infections.
Genomic Analysis Reveals Variation between
Mycobacterium tuberculosis
H37Rv and the Attenuated
M. tuberculosis
H37Ra Strain
Authors: Brosch Roland , Philipp Wolfgang J. , Stavropoulos Evangelos , Colston M. Joseph , Cole Stewart T. , Gordon Stephen V.
Published: December 2019

Mycobacterium tuberculosis
H37Ra is an attenuated tubercle bacillus closely related to the virulent type strain
M. tuberculosis
H37Rv. Despite extensive study, the reason for the decreased virulence of
M. tuberculosis
H37Ra has not been determined. A genomic approach was therefore initiated to identify genetic differences between
M. tuberculosis
H37Rv and
M. tuberculosis
H37Ra as a means of pinpointing the attenuating mutation(s). Digestion with the rare-cutting restriction endonuclease
Dra
I revealed two polymorphisms between the strains: a 480-kb fragment in
M. tuberculosis
H37Rv was replaced by two fragments of 220 and 260 kb in
M. tuberculosis
H37Ra, while there was a ∼7.9-kb
Dra
I fragment in
M. tuberculosis
H37Ra that had no counterpart in
M. tuberculosis
H37Rv. As the
M. tuberculosis
insertion sequence IS
6110
contains a single
Dra
I restriction site, it was considered possible that these polymorphisms were the result of IS
6110
transposition events in
M. tuberculosis
H37Ra, events that may have inactivated virulence genes. The 7.9-kb polymorphism was found to be due to the presence of the previously described H37Rv RvD2 deletion in
M. tuberculosis
H37Ra, with sequence analysis suggesting an IS
6110
-mediated deletion mechanism for loss of RvD2. Three other IS
6110
-catalyzed deletions from the
M. tuberculosis
H37Rv chromosome (RvD3 to RvD5) were also identified, suggesting that this mechanism plays an important role in genome plasticity in the tubercle bacilli. Comparative mapping and sequencing revealed that the 480-kb polymorphism was due to an IS
6110
insertion in
M. tuberculosis
H37Ra near
oriC
. Complementation of
M. tuberculosis
H37Ra with a 2.9-kb restriction fragment from
M. tuberculosis
H37Rv that encompassed the IS
6110
insertion did not increase the survival of recombinant
M. tuberculosis
H37Ra in mice. In conclusion, this study describes the presence and mechanisms of genomic variation between
M. tuberculosis
H37Ra and
M. tuberculosis
H37Rv, although the role that they play in the attenuation of
M. tuberculosis
H37Ra is unclear.
Native and Mutant Forms of Cholera Toxin and Heat-Labile Enterotoxin Effectively Enhance Protective Efficacy of Live Attenuated and Heat-KilledShigellaVaccines
Authors: Hartman Antoinette B. , Van De Verg Lillian L. , Venkatesan Malabi M.
Published: December 2019

Both native and mutant forms of cholera toxin (CT) and heat-labile enterotoxin (LT) are effective adjuvants for antigens and killed whole-cell preparations. To determine whether these toxin molecules could also boost the immunogenicity and efficacy of live attenuated vaccines directed against shigellosis, the guinea pig keratoconjunctivitis model was used to evaluate the adjuvant effect of these toxin molecules on EcSf2a-3, a ΔvirG ΔaroD Escherichia coli-Shigella flexneri2a hybrid vaccine strain that was previously found to be less protective than its parent strain in the guinea pig model. Experiments using native and mutant toxin molecules showed that both CT and LT and mutant derivatives were effective as an adjuvant for EcSf2a-3 and that the mutant toxin molecules, which were developed to retain adjuvanticity without the toxicity associated with the native molecules, were as effective as the native toxin molecules as adjuvants. Protective efficacy was enhanced for both the oral and intranasal routes of immunization. Serum antibody response to theS. flexneri2a O antigen, the primary antigen for protective immunity, was not dependent on the addition of an adjuvant. However, enumeration of the O-antigen-specific immunoglobulin G (IgG) and IgA antibody-secreting cells in the spleen and draining lymph nodes following intranasal immunization suggested that enhancement of the local immune response by the toxin molecules may contribute to the observed increase in protective efficacy. The efficacy of heat-killedS. flexneri2a was enhanced only by mutant LT molecules. These results suggest that the best candidates for enhancing the efficacy of both live attenuated and heat-killedShigellavaccines with minimal reactogenicity are the mutant toxin molecules.
Coadministration of Interleukin 12 Expression Vector with Antigen 2 cDNA Enhances Induction of Protective Immunity againstCoccidioides immitis
Authors: Jiang Chengyong , Magee D. Mitchell , Cox Rebecca A.
Published: December 2019

Interleukin 12 (IL-12) plays an important role in the induction of protective immunity against cancer and infectious diseases. In this study we asked whether IL-12 cDNA could increase the protective capacity of the antigen 2 (Ag2) gene vaccine in experimental coccidioidomycosis. Coimmunization of BALB/c mice with a single-chain IL-12 cDNA (p40-L-p35) and Ag2 cDNA, both subcloned into the pVR1012 plasmid, significantly enhanced protection against systemic challenge with 2,500 arthroconidia, as evidenced by a greater-than-1.3-log-unit reduction in the fungal load in the lungs and spleens compared to mice receiving the pVR1012 vector alone, Ag2 cDNA alone, or IL-12 cDNA alone. The enhanced protection was associated with increased gamma interferon secretion; production of immunoglobulin G2a (IgG2a), IgG2b, and IgG3 antibodies toCoccidioides immitisantigen; and the influx of CD4+and CD8+T cells in lungs and spleens. When challenged by the pulmonary route, mice covaccinated with Ag2 cDNA and IL-12 cDNA were not protected at the lung level but did show a significant reduction in the fungal load in their livers and spleens compared to mice vaccinated with Ag2 cDNA or IL-12 cDNA alone. These results suggest that IL-12 acts as a therapeutic adjuvant to enhance Ag2 cDNA-induced protective immunity against experimental coccidioidomycosis through the induction of Th1-associated immune responses.
Immune CD8+T Cells Prevent Reactivation ofToxoplasma gondiiInfection in the Immunocompromised Host
Authors: Khan Imtiaz A. , Green William R. , Kasper Lloyd H. , Green Kathy A. , Schwartzman Joseph D.
Published: December 2019

Toxoplasma gondiiremains a serious cause of morbidity and mortality in individuals that are immunosuppressed, patients with AIDS in particular. The cellular immune response, especially by gamma interferon (IFN-γ)-producing CD8+T cells, is an essential component of protective immunity against the parasite. In the present study the role of CD8+T cells during the reactivation ofToxoplasmainfection in an immunocompromised murine model was evaluated. Chronically infected mice were challenged with LP-BM5 virus, and the kinetics of CD8+T-cell function was studied. At 10 weeks after viral infection, mice showed obvious signs of systemic illness and began to die. At this stage, CD8+T cells were unresponsive to antigenic stimulation and unable to killToxoplasma-infected targets. IFN-γ production by the CD8+T cells from dual-infected animals reached background levels, and a dramatic fall in the frequency of precursor cytotoxic T lymphocytes was observed. Histopathological analysis of the tissues demonstrated signs of disseminated toxoplasmosis as a result of reactivation of infection. However, treatment of the dual-infected animals with immune CD8+T cells at 5 weeks post-LP-BM5 challenge prevented the reactivation of toxoplasmosis, and mice continued to live. Our study for the first time demonstrates a therapeutic role for CD8+T cells against an opportunistic infection in an immunocompromised state.
Klebsiella pneumoniae
Capsule Expression Is Necessary for Colonization of Large Intestines of Streptomycin-Treated Mice
Authors: Favre-Bonté Sabine , Licht Tine Rask , Forestier Christiane , Krogfelt Karen Angeliki
Published: December 2019

The role of the
Klebsiella pneumoniae
capsular polysaccharide (K antigen) during colonization of the mouse large intestine was assessed with wild-type
K. pneumoniae
LM21 and its isogenic capsule-defective mutant. When bacterial strains were fed alone to mice, the capsulated bacteria persisted in the intestinal tract at levels of 10
8
CFU/g of feces while the capsule-defective strain colonized at low levels, 10
4
CFU/g of feces. In mixed-infection experiments, the mutant was rapidly outcompeted by the wild type. In situ hybridization on colonic sections revealed that bacterial cells of both strains were evenly distributed in the mucus layer at day 1 after infection, while at day 20 the wild type remained dispersed and the capsule-defective strain was seen in clusters in the mucus layer. These results suggest that capsular polysaccharide plays an important role in the gut colonization ability of
K. pneumoniae
.
Characterization of a Novel Methyl-Accepting Chemotaxis Gene,
dmcB
, from the Oral Spirochete
Treponema denticola
Authors: Li Hong , Arakawa Shinichi , Deng Qing-Dong , Kuramitsu Howard
Published: December 2019

Immediately downstream from the previously isolated
Treponema denticola
ATCC 35405
prtB
gene coding for a chymotrypsinlike protease activity, an open reading frame, ORF3, was identified which shared significant homology with the highly conserved domains (HCDs) of bacterial methyl-accepting chemotaxis proteins (MCPs). Nucleotide sequencing of this ORF revealed that the gene would code for a protein with a size of approximately 41 kDa. In addition, this sequence contained a domain which was virtually identical to the HCD of a recently characterized MCP, DmcA, of strain 35405. Therefore, this ORF was named
dmcB
. Northern blot analysis suggested that
dmcB
was part of an operon structure containing
prtB
. Insertional inactivation of
dmcB
utilizing an
ermF-ermAM
cassette resulted in a mutant with decreased chemoattraction toward nutrient supplements. In addition, the mutant displayed an altered pattern of methylated proteins under conditions of chemotaxis. Inactivation of the
dmcB
gene also attenuated the methylation of the DmcA protein. These results suggest that the
dmcB
gene codes for an MCP in
T. denticola
which may interact with other MCPs in these organisms.
Effectiveness of a Vaccine Composed of Heat-KilledCandida albicansand a Novel Mucosal Adjuvant, LT(R192G), against Systemic Candidiasis
Authors: Cárdenas-Freytag Lucía , Cheng Elly , Mayeux Patricia , Domer Judith E. , Clements John D.
Published: December 2019

The incidence of fungal infections caused by the opportunistic yeastCandida albicanshas increased significantly in recent years. The ability to vaccinate selected patients against the organism would be advantageous. In this paper we describe a potential anti-C. albicansvaccine consisting of heat-killedC. albicans(HK-CA) in combination with the novel mucosal adjuvant LT(R192G), a genetically detoxified form of the heat-labile toxin of enterotoxigenicEscherichia coli. Groups of male CBA/J mice were immunized intranasally on three occasions at weekly intervals with 2 × 107HK-CA per dose, alone or in conjunction with 10 μg of LT(R192G) per dose. Two weeks following the last application of antigen, some animals were challenged intravenously (i.v.) with 104, 105, or 106viableC. albicansto assess protection as measured by survival and/or culture. Some groups of animals were footpad tested withC. albicansmannan to assess delayed-type hypersensitivity (DTH), and all the animals were bled for antibody assays. In two independent studies, all the animals immunized with HK-CA plus LT(R192G) were able to eradicate 104C. albicanscompletely, as determined by kidney culture 4 weeks after challenge. Animals immunized with HK-CA only had reduced levels ofC. albicanscompared to the adjuvant or saline-only control. Greatly enhanced survival was observed when mice immunized with HK-CA plus LT(R192G) were challenged with 105liveC. albicansas well. Animals immunized with HK-CA plus LT(R192G) developed a significant DH response, while those given HK-CA alone developed only marginal DH responses. High immunoglobulin G (IgG) levels to cytoplasmic antigens developed in mice immunized with HK-CA plus LT(R192G), but they were found only after i.v. challenge. Addition of adjuvant shifted the antibody isotype production in i.v.-challenged animals to a response dominated by IgG2a. Clearly, intranasal immunization with killedC. albicansin conjunction with LT(R192G) afforded significant levels of protection. This novel approach offers new possibilities for the development of an effective vaccine against candidiasis for use in humans.
Comparative Virulence of
Streptococcus pneumoniae
Strains with Insertion-Duplication, Point, and Deletion Mutations in the Pneumolysin Gene
Authors: Berry Anne M. , Ogunniyi A. David , Miller David C. , Paton James C.
Published: December 2019

Pneumolysin is a 471-amino-acid toxin produced by
Streptococcus pneumoniae
which has both cytolytic and complement activation properties. We have constructed a derivative of the type 2
S. pneumoniae
strain D39 in which the portion of the pneumolysin gene encoding amino acids 55 to 437 has been deleted in-frame. The virulence of this strain (ΔPly) was compared with those of wild-type D39, a pneumolysin insertion-duplication mutant (PLN-A), and a derivative (PdT) carrying a toxin gene with three point mutations known to abolish both cytolytic activity and complement activation. PdT was intermediate in virulence between D39 and either PLN-A or ΔPly in a mouse intraperitoneal challenge model. This provides unequivocal evidence that pneumolysin has an additional property that is not abolished by point mutations which reduce cytotoxicity and complement activation to virtually undetectable levels.
Enhanced Th1 and Dampened Th2 Responses Synergize To Inhibit Acute Granulomatous and Fibrotic Responses in Murine Schistosomiasis Mansoni
Authors: Boros Dov L. , Whitfield Joel R.
Published: December 2019

In murine schistosomiasis mansoni, CD4+Th1 and Th2 cells participate in the ovum-induced granulomatous inflammation. Previous studies showed that the interleukin-12 (IL-12)-induced Th1 response strongly suppressed the Th2-cell-mediated pulmonary granuloma development in naive or primed mice. However, liver granulomas were only moderately suppressed in egg-vaccinated, recombinant IL-12 (rIL-12)-treated infected mice. The present study shows that repeated rIL-12 injections given during early granuloma development at 5 to 7 weeks after infection prolonged the Th1 phase and resulted in gamma interferon-mediated suppression of liver granulomas. The timing is crucial: if given at 6 to 8 weeks, during the Th2-dominated phase of florid granuloma growth, the treatment is ineffective. Daily injections of rIL-12 given between 5 and 7.5 weeks during the period of granuloma growth achieved a somewhat-stronger diminution in granuloma growth with less deposition of collagen but caused 60% mortality and liver pathology. In contrast, combined treatment with rIL-12 and anti-IL-4–anti-IL-10 monoclonal antibody (MAb) injections given during the Th2 phase strongly inhibited liver granuloma growth without mortality. The diminished inflammatory response was accompanied by less deposition of collagen in the liver. Moreover, neutralization of endogenous IL-12 by anti-IL-12 MAbs effectively decreased the early Th1 phase (between 5 and 6 weeks after infection) but not the developing Th2 phase (5 to 7 weeks) of granuloma development. These studies indicate that the granulomatous response in infected mice can be manipulated by utilizing the Th1-Th2-subset antagonism with potential salutary results in the amelioration of fibrous pathology.
Zonula Occludens Toxin Is a Powerful Mucosal Adjuvant for Intranasally Delivered Antigens
Authors: Marinaro Mariarosaria , di Tommaso Annalisa , Uzzau Sergio , Fasano Alessio , de Magistris Maria Teresa
Published: December 2019

Zonula occludens toxin (Zot) is produced by toxigenic strains ofVibrio choleraeand has the ability to reversibly alter intestinal epithelial tight junctions, allowing the passage of macromolecules through the mucosal barrier. In the present study, we investigated whether Zot could be exploited to deliver soluble antigens through the nasal mucosa for the induction of antigen-specific systemic and mucosal immune responses. Intranasal immunization of mice with ovalbumin (Ova) and recombinant Zot, either fused to the maltose-binding protein (MBP-Zot) or with a hexahistidine tag (His-Zot), induced anti-Ova serum immunoglobulin G (IgG) titers that were approximately 40-fold higher than those induced by immunization with antigen alone. Interestingly, Zot also stimulated high anti-Ova IgA titers in serum, as well as in vaginal and intestinal secretions. A comparison withEscherichia coliheat-labile enterotoxin (LT) revealed that the adjuvant activity of Zot was only sevenfold lower than that of LT. Moreover, Zot and LT induced similar patterns of Ova-specific IgG subclasses. The subtypes IgG1, IgG2a, and IgG2b were all stimulated, with a predominance of IgG1 and IgG2b. In conclusion, our results highlight Zot as a novel potent mucosal adjuvant of microbial origin.
Cryptosporidium parvumSporozoite Pellicle Antigen Recognized by a Neutralizing Monoclonal Antibody Is a β-Mannosylated Glycolipid
Authors: Riggs Michael W. , McNeil Michael R. , Perryman Lance E. , Stone Alice L. , Scherman Michael S. , O’Connor Roberta M.
Published: December 2019

The protozoan parasiteCryptosporidium parvumis an important cause of diarrhea in humans, calves, and other mammals worldwide. No approved vaccines or parasite-specific drugs are currently available for the control of cryptosporidiosis. To effectively immunize againstC. parvum, identification and characterization of protective antigens are required. We previously identified CPS-500, a conserved, neutralization-sensitive antigen ofC. parvumsporozoites and merozoites defined by monoclonal antibody 18.44. In the present study, the biochemical characteristics and subcellular location of CPS-500 were determined. CPS-500 was chloroform extractable and eluted with acetone and methanol in silicic acid chromatography, consistent with being a polar glycolipid. Following chloroform extraction and silicic acid chromatography, CPS-500 was isolated by high-pressure liquid chromatography for glycosyl analysis, which indicated the presence of mannose and inositol. To identify which component of CPS-500 comprised the neutralization-sensitive epitope recognized by 18.44, the ability of the monoclonal antibody to bind CPS-500 treated with proteases, or with α- or β-glycosidases, was determined. Monoclonal antibody 18.44 did not bind antigen treated with β-d-mannosidase but did bind antigen treated with α-d-mannosidase, other α- or β-glycosidases, or a panel of proteases. These data indicated that the target epitope was dependent on terminal β-d-mannopyranosyl residues. By immunoelectron microscopy, 18.44 binding was localized to the pellicle and an intracytoplasmic tubulovesicular network in sporozoites. Monoclonal antibody 18.44 also bound to antigen deposited and released onto substrate over the course travelled by gliding sporozoites and merozoites. Surface localization, adhesion and release during locomotion, and neutralization sensitivity suggest that CPS-500 may be involved in motility and invasion processes of the infective zoite stages.
The Virulence Plasmid-Encoded
impCAB
Operon Enhances Survival and Induced Mutagenesis in
Shigella flexneri
after Exposure to UV Radiation
Authors: Runyen-Janecky Laura J. , Hong Mei , Payne Shelley M.
Published: December 2019

Upon exposure to UV radiation,
Shigella flexneri
SA100 displayed survival and mutation frequencies comparable to those of
Escherichia coli
AB1157, which contains a functional UmuDC error-prone DNA repair system. Survival of SA100 after UV irradiation was associated with the presence of the 220-kb virulence plasmid, pVP. This plasmid encodes homologues of ImpA and ImpB, which comprise an error-prone DNA repair system encoded on plasmid TP110 that was initially identified in
Salmonella typhimurium
, and ImpC, encoded upstream of ImpA and ImpB. Although the
impB
gene was present in representatives of all four species of
Shigella
, not all isolates tested contained the gene.
Shigella
isolates that lacked
impB
were more sensitive to UV radiation than isolates that contained
impB
. The nucleotide sequence of a 2.4-kb DNA fragment containing the
imp
operon from
S. flexneri
SA100 pVP was 96% identical to the
imp
operon from the plasmid TP110. An SA100 derivative with a mutation in the
impB
gene had reduced survival following UV irradiation and less UV-induced mutagenesis relative to the parental strain. We also found that
S. flexneri
contained a chromosomally encoded
umuDC
operon; however, the
umuDC
promoter was not induced by exposure to UV radiation. This suggests that the
imp
operon but not the
umuDC
operon contributes to survival and induced mutagenesis in
S. flexneri
following exposure to UV radiation.
Lipopolysaccharide (LPS) from
Burkholderia cepacia
Is More Active than LPS from
Pseudomonas aeruginosa
and
Stenotrophomonas maltophilia
in Stimulating Tumor Necrosis Factor Alpha from Human Monocytes
Authors: Zughaier Susu M. , Ryley Henry C. , Jackson Simon K.
Published: December 2019

Whole cells and lipopolysaccharides (LPSs) extracted from
Burkholderia cepacia
,
Pseudomonas aeruginosa
,
Stenotrophomonas maltophilia
, and
Escherichia coli
were compared in their ability to stimulate tumor necrosis factor alpha (TNF-α) from the human monocyte cell line MonoMac-6.
B. cepacia
LPS, on a weight-for-weight basis, was found to have TNF-α-inducing activity similar to that of LPS from
E. coli
, which was approximately four- and eightfold greater than the activity of LPSs from
P. aeruginosa
and
S. maltophilia
, respectively. The LPS-stimulated TNF-α production from monocytes was found to be CD14 dependent. These results suggest that
B. cepacia
LPS might play a role in the pathogenesis of inflammatory lung disease in cystic fibrosis, and in some patients it might be responsible, at least in part, for the sepsis-like cepacia syndrome.
Effects of Polysaccharide Fucoidin on Cerebrospinal Fluid Interleukin-1 and Tumor Necrosis Factor Alpha in Pneumococcal Meningitis in the Rabbit
Authors: Granert Carl , Raud Johan , Waage Anders , Lindquist Lars
Published: December 2019

The inflammatory response in bacterial meningitis is mediated by cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin-1 (IL-1), which are produced in the subarachnoid space by different cells, e.g., leukocytes, astrocytes, and microglia. The recruitment of leukocytes into the cerebrospinal fluid (CSF) has been shown to contribute to the neurological damage in this disease, a process which could be enhanced by treatment with antibiotics. In this study, we have used a rabbit meningitis model for two sets of experiments with intracisternal (i.c.) injections ofStreptococcus pneumoniae. First, pneumococcal cell wall (PCW) components were injected i.c., inducing an inflammatory response with pleocytosis and increased levels of CSF TNF-α) and IL-1 at 6 and 12 h after PCW injection. Treatment with fucoidin, known to inhibit leukocyte rolling, abolished pleocytosis and inhibited the release of TNF-α and IL-1. In the second experiment, live pneumococcal bacteria were injected i.c. and treatment with one dose of ampicillin (40 mg/kg of body weight intravenously) was given 16 h after induction of meningitis, causing a sevenfold increase in CSF leukocytes over a 4-h period. CSF IL-1 levels at 16 h were high but did not increase further at 20 h. Also, CSF TNF-α levels were high at 16 h and tended to increase at 20 h. Fucoidin treatment prevented the antibiotic-induced increase of CSF leukocytes but had no effect on the TNF-α and IL-1 levels. Taken together, fucoidin reduced CSF TNF-α and IL-1 levels in acute bacterial meningitis induced by PCW fragments but had no effect later in the course of the disease, when live bacteria were used and an inflammatory increase was caused by a dose of antibiotics.
Immune Responses Induced in Cattle by Virulent and Attenuated
Mycobacterium bovis
Strains: Correlation of Delayed-Type Hypersensitivity with Ability of Strains To Grow in Macrophages
Authors: Wedlock D. Neil , Aldwell Frank E. , Collins Desmond M. , de Lisle Geoffrey W. , Wilson Theresa , Buddle Bryce M.
Published: December 2019

Comparison of immune responses induced in cattle by virulent and attenuated strains of
Mycobacterium bovis
will assist in identifying responses associated with resistance or susceptibility to disease. Four strains of
M. bovis
, one which is virulent in guinea pigs (WAg201) and three which are attenuated in guinea pigs (an isoniazid-resistant strain [WAg405], ATCC 35721, and BCG) were compared for their abilities to induce immune responses in cattle and to grow in bovine lung alveolar macrophage cultures. Extensive macroscopic lesions were found only in cattle inoculated with the virulent
M. bovis
strain. Strong antibody responses to
M. bovis
culture filtrate, as well as persistently high levels of gamma interferon and interleukin-2 released from purified protein derivative (PPD)-stimulated peripheral blood lymphocyte cultures, were observed in the cattle inoculated with the virulent strain compared to those inoculated with the attenuated strains. All cattle inoculated with the virulent strain or two of the attenuated strains (WAg405 and ATCC 35721) elicited strong delayed-type hypersensitivity responses to PPD in skin tests, while animals inoculated with BCG induced only a weak response. The three strains which produced strong skin test responses proliferated well in bovine alveolar macrophages and induced high levels of proinflammatory cytokine mRNAs compared to BCG. Our study showed that skin test responsiveness to PPD correlated with the ability of the strains to grow in alveolar macrophages rather than to their pathogenicity in cattle.