subsp.
serovar Gallinarum (SG) is a common pathogen in chickens, and causes an acute systemic disease that leads to high mortality. The live attenuated vaccine 9R is able to successfully protect ...chickens older than six weeks by activating a robust cell-mediated immune response, but its safety and efficacy in young chickens remains controversial. An inactivated SG vaccine is being used as an alternative, but because of its low cellular immune response, it cannot be used as a replacement for live attenuated 9R vaccine. In this study, we employed gamma irradiation instead of formalin as an inactivation method to increase the efficacy of the inactivated SG vaccine. Humoral, cellular, and protective immune responses were compared in both mouse and chicken models. The radiation-inactivated SG vaccine (r-SG) induced production of significantly higher levels of IgG2b and IgG3 antibodies than the formalin-inactivated vaccine (f-SG), and provided a homogeneous functional antibody response against group D, but not group B Salmonella. Moreover, we found that r-SG vaccination could provide a higher protective immune response than f-SG by inducing higher Th17 activation. These results indicate that r-SG can provide a protective immune response similar to the live attenuated 9R vaccine by activating a higher humoral immunity and a lower, but still protective, cellular immune response. Therefore, we expect that the radiation inactivation method might substitute for the 9R vaccine with little or no side effects in chickens younger than six weeks.
is an extremely resistant bacterium against extracellular stress owing to on its unique physiological functions and the structure of its cellular constituents. Interestingly, it has been reported ...that the pattern of alteration in
proportion on the skin is negatively correlated with skin inflammatory diseases, whereas the proportion of
was increased in patients with chronic skin inflammatory diseases. However, the biological mechanisms of deinococcal interactions with other skin commensal bacteria have not been studied. In this study, we hypothesized that deinococcal cellular constituents play a pivotal role in preventing
colonization by inhibiting biofilm formation. To prove this, we first isolated cellular constituents, such as exopolysaccharide (DeinoPol), cell wall (DeinoWall), and cell membrane (DeinoMem), from
and investigated their inhibitory effects on
colonization and biofilm formation
and
. Among them, only DeinoPol exhibited an anti-biofilm effect without affecting bacterial growth and inhibiting staphylococcal colonization and inflammation in a mouse skin infection model. Moreover, the inhibitory effect was impaired in the Δ
strain, a mutant that cannot produce DeinoPol. Remarkably, DeinoPol not only interfered with
biofilm formation at early and late stages but also disrupted a preexisting biofilm by inhibiting the production of poly-
-acetylglucosamine (PNAG), a key molecule required for
biofilm formation. Taken together, the present study suggests that DeinoPol is a key molecule in the negative regulation of
biofilm formation by
. Therefore, DeinoPol could be applied to prevent and/or treat infections or inflammatory diseases associated with
biofilms.
Recently, respiratory syncytial virus (RSV) vaccines based on the prefusion F (pre-F) antigen were approved in the United States. We aimed to develop an enzyme-linked immunosorbent assay ...(ELISA)-based protocol for the practical and large-scale evaluation of RSV vaccines. Two modified pre-F proteins (DS-Cav1 and SC-TM) were produced by genetic recombination and replication using an adenoviral vector. The protocol was established by optimizing the concentrations of the coating antigen (pre-F proteins), secondary antibodies, and blocking buffer. To validate the protocol, we examined its accuracy, precision, and specificity using serum samples from 150 participants across various age groups and the standard serum provided by the National Institute of Health. In the linear correlation analysis, coating concentrations of 5 and 2.5 μg/mL of DS-Cav1 and SC-TM showed high coefficients of determination (r > 0.90), respectively. Concentrations of secondary antibodies (alkaline phosphatase-conjugated anti-human immunoglobulin G, diluted 1:2000) and blocking reagents (5% skim milk/PBS-T) were optimized to minimize non-specific reactions. High accuracy was observed for DS-Cav1 (r = 0.90) and SC-TM (r = 0.86). Further, both antigens showed high precision (coefficient of variation < 15%). Inhibition ELISA revealed cross-reactivity of antibodies against DS-Cav1 and SC-TM, but not with the attachment (G) protein.
Expression of bacteriophage lysin
by Streptococcus oralis strain SF100 is thought to be important for the pathogenesis of infective endocarditis, due to its ability to mediate bacterial binding to ...fibrinogen. To better define the lysin
binding site on fibrinogen Aα, and to investigate the impact of binding on fibrinolysis, we examined the interaction of lysin
with a series of recombinant fibrinogen Aα variants. These studies revealed that lysin
binds the C-terminal region of fibrinogen Aα spanned by amino acid residues 534 to 610, with an affinity of equilibrium dissociation constant (
) of 3.23 × 10
M. This binding site overlaps the known binding site for plasminogen, an inactive precursor of plasmin, which is a key protease responsible for degrading fibrin polymers. When tested
, lysin
competitively inhibited plasminogen binding to the αC region of fibrinogen Aα. It also inhibited plasminogen-mediated fibrinolysis, as measured by thromboelastography (TEG). These results indicate that lysin
is a bi-functional virulence factor for streptococci, serving as both an adhesin and a plasminogen inhibitor. Thus, lysin
may facilitate the attachment of bacteria to fibrinogen on the surface of damaged cardiac valves and may also inhibit plasminogen-mediated lysis of infected thrombi (vegetations) on valve surfaces.
The interaction of streptococci with human fibrinogen and platelets on damaged endocardium is a central event in the pathogenesis of infective endocarditis. Streptococcus oralis can bind platelets via the interaction of bacteriophage lysin
with fibrinogen on the platelet surface, and this process has been associated with increased virulence in an animal model of endocarditis. We now report that lysin
binds to the αC region of the human fibrinogen Aα chain. This interaction blocks plasminogen binding to fibrinogen and inhibits fibrinolysis.
, this inhibition could prevent the lysis of infected vegetations, thereby promoting bacterial persistence and virulence.
Abstract The expression of arginases, enzymes that catalyze the hydrolysis of arginine to ornithine and urea, was studied in the inflammatory lesions of spinal cord injury (SCI) in rats. The level of ...arginase-1 expression in rat spinal cords with clip compression injury was determined by Western blot analysis and immunohistochemistry. Western blot showed that the level of arginase-1 increased in the core lesion of SCI at day 1 post injury and continued to increase through days 4 ( p < 0.05) and 7 ( p < 0.01). Immunohistochemical analysis showed that arginase-1 was constitutively expressed in neurons and glial cells in sham control spinal cords. In SCI lesions, arginase-1 was additionally detected in inflammatory cells, particularly in isolectin B4-positive macrophages and reactive astrocytes within the core lesion. These findings suggest that the increased level of arginase-1 in SCI is associated with an increase in macrophages and reactive astrocytes, possibly contributing to the modulation of inflammation during the course of SCI.
Initiation and progression of oral infectious diseases are associated with streptococcal species. Bacterial infection induces inflammatory responses together with reactive oxygen species (ROS), often ...causing cell death and tissue damage in the host. In the present study, we investigated the effects of oral streptococci on cytotoxicity and ROS production in human periodontal ligament (PDL) cells.
Streptococcus gordonii
showed cell cytotoxicity in a dose- and time-dependent manner. The cytotoxicity might be due to apoptosis since
S. gordonii
increased annexin V-positive cells, and the cytotoxicity was reduced by an apoptosis inhibitor, Z-VAD-FMK. Other oral streptococci such as
Streptococcus mitis
,
Streptococcus sanguinis
, and
Streptococcus sobrinus
also induced apoptosis, whereas
Streptococcus mutans
did not. All streptococci tested except
S. mutans
triggered ROS production in human PDL cells. Interestingly, however, streptococci-induced apoptosis appears to be ROS-independent, as the cell death induced by
S. gordonii
was not recovered by the ROS inhibitor, resveratrol or
n
-acetylcysteine. Instead, hydrogen peroxide (H
2
O
2
) appears to be important for the cytotoxic effects of streptococci since most oral streptococci except
S. mutans
generated H
2
O
2
, and the cytotoxicity was dramatically reduced by catalase. Furthermore, streptococcal lipoproteins are involved in cytotoxicity, as we observed that cytotoxicity induced by the lipoprotein-deficient
S. gordonii
mutant was less potent than that by the wild-type and was attenuated by anti-TLR2-neutralizing antibody. Indeed, lipoproteins purified from
S. gordonii
alone were sufficient to induce cytotoxicity. Notably,
S. gordonii
lipoproteins did not induce H
2
O
2
or ROS but cooperatively induced cell death when co-treated with H
2
O
2
. Taken together, these results suggest that most oral streptococci except
S. mutans
efficiently induce damage to human PDL cells by inducing apoptotic cell death with bacterial H
2
O
2
and lipoproteins, which might contribute to the progression of oral infectious diseases such as apical periodontitis.
Lipoteichoic acid (LTA) of Gram-positive bacteria is regarded as the counterpart biomolecule of lipopolysaccharide (LPS) of Gram-negative bacteria because of their structural and immunological ...similarities. Although LPS induces a strong polyclonal expansion of B cells, little is known about the effect of LTA on B cell proliferation. In the present study, we prepared LTAs from Gram-positive bacteria and examined their effect on splenic B cell proliferation. Unlike LPS, LTA did not induce B cell proliferation. Instead, Staphylococcus aureus LTA (Sa.LTA) appeared to inhibit LPS-induced B cell proliferation in vitro, ex vivo, and in vivo models. Such effect was observed neither in splenocytes from Toll-like receptor 2 (TLR2)-deficient mice nor in the purified splenic B cells. Furthermore, decreased ERK phosphorylation appeared to be responsible for this phenomenon. Collectively, our results support that Sa.LTA inhibited LPS-induced B cell proliferation through the decrease of ERK phosphorylation via TLR2 signaling pathway.
Abstract To investigate whether fibronectin, a high-molecular weight glycoprotein of the extracellular matrix (ECM), plays a role in the activation of microglia/macrophages after brain injury, we ...examined the changes in fibronectin and arginase-1, a marker for alternatively activated macrophages, in a rat cryoinjury model using Western blot analysis, real-time reverse transcription PCR and immunohistochemistry. The protein and mRNA level of fibronectin and arginase-1 significantly increased in the injury site of the ipsilateral cerebral cortex at days 4 and 7 after cryoinjury but was decreased at day 14. The immunohistochemical analysis revealed fibronectin expression in ED1-positive microglia/macrophages and reactive astrocytes, in the lesion core and periphery, respectively. Fibronectin immunoreactivity in the lesion was similar to arginase-1 except that fibronectin was detected in the ECM after cryoinjury. The present results suggest that fibronectin was extravasated into injured brain lesions via an impaired blood–brain barrier and stimulated glial cells including microglia and infiltrating macrophages in the lesion core and periphery to become alternatively activated microglia/macrophages, which modulated CNS inflammation after brain injury.
The aim of this study was to assess the antifungal efficacy of a synthetic human beta-defensin-3-C15 peptide (HBD3-C15) in Candida albicans–infected human root dentin.
Standardized root dentin blocks ...were prepared (6-mm thick, 0.7-mm-wide canal) from single-rooted human permanent premolars and infected with C. albicans for 3 weeks. They were randomly divided into 4 groups (n = 8/group), and their canals were filled with calcium hydroxide (CH), HBD3-C15 peptide, or chlorhexidine digluconate (CHX, 2%) as disinfectants or saline as control. After 1 week of disinfection, dentinal debris were harvested at depths of 200 and 400 μm from the canal lumen, and incubated in Yeast broth for 72 hours at 37°C. Then, colony-forming units (CFU) were measured to assess the antifungal efficacy of each medicament and analyzed statistically.
All medicaments showed significantly lower CFU than saline (P < .05), and their antifungal efficacies were similar at both 200- and 400-μm tubular depths (P > .05). HBD3-C15 had similar antifungal efficacy to that of CHX at both depths (P > .05), and both medicaments had significantly lower CFU than CH at both depths (P < .05).
In this ex vivo model of C. albicans–infected human root dentin, the antifungal efficacy of synthetic HBD3-C15 was comparable with CHX.
•Candida albicans biofilm is closely related to initiation and persistence of refractory apical periodontitis.•Antifungal efficacy of various intracanal medicaments were assessed in C. albicans-infected human root dentin.•In C. albicans-infected human dentin blocks, root canals were covered by mature biofilms with tubular penetration of 400 μm.•In this standardized ex vivo model, chlorhexidine and synthetic human β-defensin-3-C15 peptide were similar and highly effective at reducing C. albicans CFU, whereas calcium hydroxide had limited efficacy.
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