Molecular characterization of pneumococcal surface protein K, a potential pneumococcal vaccine antigen.

The pneumococcal capsule is indispensable for pathogenesis in systemic infections; however, many pneumococcal diseases, including conjunctivitis, otitis media, and some systemic infections in immunocompromised patients, are caused by nonencapsulated Streptococcus pneumoniae (NESp). Null capsule clade 1 (NCC1), found in group 2 NESp, expresses pneumococcal surface protein K (PspK) and is becoming prevalent among pneumococcal organisms owing to the widespread use of pneumococcal conjugate vaccines. Despite its clinical importance, the molecular mechanisms underlying the prevalence of NCC1 have not been fully elucidated. Here, we investigated the role of the R3 domain of PspK in the epithelial cell adherence of NCC1. We found that the R3 domain of PspK mediated NCC1 adherence via its direct interaction with the epithelial surface protein annexin A2. Additionally, neutralization with purified recombinant PspK-R3 or rabbit anti-UD:R3 IgG inhibited binding of NESp to lung epithelial cells in vitro. Immunization with the 'repeat' domain of PspK-R3 or PspK-UD:R3 effectively elicited mucosal and systemic immune responses against PspK-R3 and provided protection against nasopharyngeal, lung, and middle ear colonization of NESp in mice. Additionally, we found that rabbit anti-UD:R3 IgG bound to PspC-R1 of the encapsulated TIGR4 strain and that UD:R3 immunization provided protection against nasopharyngeal and lung colonization of TIGR4 and deaths by TIGR4 and D39 in mice. Further studies using 68 pneumococcal clinical isolates showed that 79% of clinical isolates showed cross-reactivity to rabbit anti-UD:R3 IgG. About 87% of serotypes in the 13-valent pneumococcal conjugate vaccine (PCV) and 68% of non-vaccine serotypes were positive for cross-reactivity with rabbit anti-UD:R3 IgG. Thus, the R3 domain of PspK may be an effective vaccine candidate for both NESp and encapsulated Sp.

Improved cell viability of biocompatible nutrient-contained polymeric nanofibers.

Nanofibers containing cell nutrients (PGDs) were fabricated by mixing 5 wt% poly(epsilon-caprolactone) (P), 4 wt% gelatin (G), and 0-2.4 wt% Dulbecco's Modified Eagle's Medium (D). The contact angles showed a considerable decrease from 118.4 degrees on the P scaffold to 17.6 degrees on PGD1.6 (containing 1.6 wt% D). The weight loss ratios between PGD1.6 and the P nanofiber, and between PGD1.6 and the PG nanofiber by degradation after 28 days were approximately 3.1 and 1.4, respectively. The rate of cell proliferation on PGD1.6 was greater than that on the PG nanofiber by 14% and 38% for the exchanged and unexchanged culture media, respectively. The physicochemical measurement results showed that the PGDs exhibited enhanced hydrophilic properties and rapid biodegradation. The PGD nanofibers with increasing D content showed better conditions for long-term cell viability. The growth mechanism of the cells on the PGDs was explained by an attachment and growth process.

Cross-linking of MHC class II molecules interferes with phorbol 12,13-dibutyrate-induced differentiation of resting B cells by inhibiting Rac-associated ROS-dependent ERK/p38 MAP kinase pathways leading to NF-kappaB activation.

In addition to their essential role in antigen presentation, major histocompatibility complex (MHC) class II molecules have been described as the receptor associated with signal transduction regulating B-cell function. In previous experiments, we found that cross-linking of MHC class II molecules with corresponding anti-MHC class II antibodies inhibited NF-kappaB-activated signaling pathways associated with the proliferation and differentiation of the LPS-stimulated primary and resting B-cell line, 38B9. We also found that exposure to the anti-MHC class II antibody reduced the production of ROS, which function as secondary signal transducers, in the phorbol 12,13-dibutyrate (PDBU)-treated (but not in the LPS-treated) resting B-cell line. In this study, we investigated the molecular mechanisms in the ROS-associated signaling pathway leading to PDBU-induced NF-kappaB activation that results in B-cell differentiation and speculated that the signaling pathway was inhibited by exposure to the anti-MHC class II antibody. We also found that this inhibition was mediated through down-regulation of the activated Rac/ROS-associated ERK/p38 MAPK signaling pathway in PDBU-treated 38B9 cells. Collectively, these findings suggest that ROS-associated molecules are involved in MHC class II-associated negative signal transduction in resting B cells.

Induction of protective immune responses against the challenge of Actinobacillus pleuropneumoniae by the oral administration of transgenic tobacco plant expressing ApxIIA toxin from the bacteria.

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia. Among the virulence factors, ApxIIA, a bacterial exotoxin, is reportedly expressed in many serotypes and is considered as a candidate for the development of a vaccine against the bacterial infection. Previously, we isolated a field strain of A. pleuropneumoniae serotype 2 in Korea and characterized its exotoxins to develop an oral vaccine. In this study, we initially confirmed the immunogenicity of ApxIIA expressed in Escherichia coli. We then developed transgenic tobacco expressing ApxIIA and tested its efficacy to induce a protective immune response against A. pleuropneumoniae infection after oral administration of the plant powder. We observed that protective immune responses were induced in mice after oral administration of the plant powder once a week for 4 weeks. Immunoassays revealed that the levels of antigen-specific immunoglobulin G against ApxIIA increased in mice that were fed a powder made from the transgenic plant, but not in mice fed a powder made from wild-type tobacco. Additionally, mice fed the transgenic plant powder were protected from an injection of a lethal dose of A. pleuropneumoniae. These results support that the transgenic plant may be a suitable candidate for an oral vaccine that could be used effectively against A. pleuropneumoniae infection.

Germinal center-independent affinity maturation in tumor necrosis factor receptor 1-deficient mice.

Germinal centers (GCs) have been identified as site at which the somatic mutation of immunoglobulins occurs.However, somatic mutations in immunoglobulins have also been observed in animals that normally do not harbor germinal centers. This clearly indicates that somatic mutations can occur in the absence of germinal centers. We therefore attempted to determine whether or not GCs exist in TNFR1-deficient mice, and are essential for the somatic mutation of immunoglobulins, using (4-hydroxy-3-nitropheny)acetyl-ovalbumin (NP-OVA). Both wild-type and TNFR1-deficient mice were immunized with NPOVA, and then examined with regard to the existence of GCs. No typical B-cell follicles were detected in the TNFR1-deficient mice. Cell proliferation was detected throughout all splenic tissue types, and no in vivo immunecomplex retention was observed in the TNFR1-deficient mice. All of these data strongly suggest that no GCs were formed in the TNFR1-deficient mice. Although TNFR1-deficient mice are unable to form GCs, serological analyses indicated that affinity maturation had been achieved in both the wild-type and TNFR1-deficient mice. We therefore isolated and sequenced several DNA clones from wild-type and the TNFR1-deficient mice. Eight out of 12 wild-type clones, and 11 out of 14 clones of the TNFR-1-deficient mice contained mutations at the CDR1 site. Thus, the wild-type and TNFR1-deficient mice were not extremely different with regard to types and rates of somatic mutation. Also, high-affinity antibodies were detected in both types of mice. Collectively, our data appear to show that affinity maturation may occur in TNFR1-deficient mice, which completely lack GCs.

Lipoteichoic acid-induced nitric oxide production depends on the activation of platelet-activating factor receptor and Jak2.

NO production by macrophages in response to lipoteichoic acid (LTA) and a synthetic lipopeptide (Pam3CSK4) was investigated. LTA and Pam3CSK4 induced the production of both TNF-alpha and NO. Inhibitors of platelet-activating factor receptor (PAFR) blocked LTA- or Pam3CSK4-induced production of NO but not TNF-alpha. Jak2 tyrosine kinase inhibition blocked LTA-induced production of NO but not TNF-alpha. PAFR inhibition blocked phosphorylation of Jak2 and STAT1, a key factor for expressing inducible NO synthase. In addition, LTA did not induce IFN-beta expression, and p38 mitogen-activated protein serine kinase was necessary for LTA-induced NO production but not for TNF-alpha production. These findings suggest that Gram-positive bacteria induce NO production using a PAFR signaling pathway to activate STAT1 via Jak2. This PAFR/Jak2/STAT1 signaling pathway resembles the IFN-beta, type I IFNR/Jak/STAT1 pathway described for LPS. Consequently, Gram-positive and Gram-negative bacteria appear to have different but analogous mechanisms for NO production.

Quercetin, a bioflavonoid, accelerates TNF-alpha-induced growth inhibition and apoptosis in MC3T3-E1 osteoblastic cells.

The bioflavonoid quercetin is believed to play an important role in preventing bone loss by affecting osteoclastogenesis and regulating many systemic and local factors including hormones and cytokines. This study examined how quercetin acts on tumor necrosis factor-alpha (TNF-alpha)-mediated growth inhibition and apoptosis in MC3T3-E1 osteoblastic cells. Tritium uptake assay showed that a quercetin treatment accelerated TNF-alpha-induced inhibition of DNA synthesis in the cells in a dose-dependent manner. Both the 3-(4,5-dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide and trypan blue staining assays also showed the quercetin-mediated facilitation of TNF-alpha-induced cytotoxicity in the cells. Apoptosis assays revealed an accelerating effect of quercetin on TNF-alpha-induced apoptosis in MC3T3-E1 cells. In addition, Fas activation and poly (ADP ribose) polymerase cleavage are thought to be closely associated with the TNF-alpha-induced apoptosis and its acceleration by the quercetin treatment in the cells. Collectively, this study showed that quercetin accelerates the TNF-alpha-induced growth inhibition and apoptosis in MC3T3-E1 osteoblastic cells.

Antioxidant property of an active component purified from the leaves of paraquat-tolerant Rehmannia glutinosa.

Acteoside extracted from the leaves of Rehmannia glutinosa was examined to determine the mechanism(s) of its antioxidant properties. The deoxyribose assay system showed that acteoside has a high redox potential as electron donor, which generates hydroxyl radicals in an Fe3+-dependent manner similar to ascorbic acid. However, the antioxidant properties of acteoside differ from those of ascorbic acid in that the superoxide anion-mediated reduction of nitroblue tetrazolium was actively inhibited by acteoside but not by ascorbic acid. Acteoside protected cells against glucose oxidase-mediated cytotoxicity and apoptosis in a dose-dependent manner. In addition, acteoside had immune stimulating effects, as shown by the acteoside-mediated increase in the level of DNA synthesis, viability, and cytokine secretion in mouse splenocytes. Moreover, acteoside inhibited the gelatinolytic activity of MMP proteins in a dose-dependent manner. Considering these results and the fact that acteoside is a water-soluble natural product, acteoside might have potential as a preventative treatment for oxidative stress-mediated diseases and have possibilities in the cosmetic industry.

Latent membrane protein 1 of Epstein-Barr virus plays an important role in the serum starvation resistance of Epstein-Barr virus-immortalized B lymphocytes.

We have previously shown that SNU-1103, which is a latency type III Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line (LCL) that was developed from a Korean cancer patient, resists serum starvation-induced G(1) arrest. In this study, we examined the role of latent membrane protein-1 (LMP-1) in serum starvation resistance, since LMP-1 is known to be essential for EBV-mediated immortalization of human B lymphocytes. The LMP-1 gene from SNU-1103 was introduced into the EBV-negative BJAB cell line, and shown to be associated with resistance to G(1) arrest during serum starvation. Western blot analyses of the LMP-1-transfected cells revealed several protein alterations as compared to vector-transfected control cells. The expression of key cell-cycle regulatory proteins was affected in the G(1) phase: the expression of cyclin D3, CDK2, p27, and E2F-4 was up-regulated, and the expression of cyclin D2, CDK6, p21, and p103 was down-regulated during serum starvation. These results imply that of the several EBV viral genes expressed in EBV-negative B lymphoma cells, LMP-1 mediates resistance to serum starvation-induced G(1) arrest. However, we cannot rule out the possibility that other EBV genes are also involved in the cell-cycle progression of the EBV-transformed LCL during serum starvation, since the altered protein expression profile of the LMP-1 transfectants was distinct from that of the SNU-1103 cells that expressed all of the EBV viral proteins.

The antioxidant, rather than prooxidant, activities of quercetin on normal cells: quercetin protects mouse thymocytes from glucose oxidase-mediated apoptosis.

The bioflavonoid quercetin is a dietary anticancer chemical that is capable of inducing apoptosis in tumor cells. Although the activity of quercetin is believed to be due to its antioxidative properties, it has recently been suggested that quercetin also has prooxidant activities, which might effect cytotoxicity directly. In this study, we used mouse thymocytes to investigate whether quercetin behaved as a protector against oxidative stress or as a cytotoxic agent. Quercetin treatment did not induce oxidative damage, but protected mouse thymocytes from glucose oxidase (GO)-mediated apoptosis in a dose-dependent manner. Furthermore, electrophoretic mobility shift assays revealed that quercetin (50 microM) treatment suppressed the GO-mediated DNA binding activity of redox state-sensitive transcription factors, such as NF-kappaB, AP-1, and p53. This result suggests that quercetin has antioxidative effects on thymocytes. More interestingly, quercetin treatment alone (50 microM) increased the DNA-binding activity of AP-1, which consisted of heterodimer of c-Jun and Fra-2. Finally, the antioxidant activity of quercetin was confirmed using a cell-free system of radical generation. Our findings suggest that quercetin protects mouse thymocytes from oxidative stress-mediated apoptosis and modulates the intracellular redox state through its antioxidant activity.

Selective priming of Th1-mediated antigen-specific immune responses following oral administration of mixed prescriptions of traditional Korean medicines.

BACKGROUND: In previous studies, we showed that oral administration of traditional Korean medicines, Soamsan (SA) and Bo-yang-hwan-o-tang (BHT), modulated antigen-specific immune responses in mice. METHODS: We attempted to strengthen cell-mediated immune responses in mice using two mixed prescriptions composed mainly of components used in SA and/or BHT. The effect of oral administration of the medicines on the induction of antigen-specific immune responses was investigated using hen egg-white lysozyme (HEL) as a model antigen system. RESULTS: Following oral administration, HEL-specific cellular immune responses were enhanced in HEL low-responder mice, and the concentrations of gamma interferon (IFN-gamma), but not interleukin (IL)-4, increased significantly. In addition, the prescriptions decreased the level of HEL-specific antibodies of the immunoglobulin (Ig)G1 subtype, which is associated with helper T lymphocyte (Th2) cell stimulation. Moreover, the presence of the medicines in vitro significantly increased IFN-gamma production from mouse splenocytes, and the magnitude of the increase was closely associated with glycoprotein concentrations. CONCLUSIONS: The Korean prescriptions enhanced anti-HEL-specific cellular immune responses by selectively priming specific subtypes of the helper T cell population. Consequently, they might be useful therapy for patients who need enhanced Th1, or to suppress Th2 immune responses.

Identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus.

In order to identify the neutralizing epitope of the porcine epidemic diarrhea virus (PEDV), the spike protein region that is presumed to contain the virus-neutralizing epitope was determined. This was based on the sequence information for the neutralizing epitope of the transmissible gastroenteritis virus (TGEV). A recombinant protein that corresponds to the spike region of TGEV was produced, and polyclonal antisera were generated using the recombinant protein. It was discovered that polyclonal antisera significantly inhibited plaque formation by PEDV, suggesting that this region of the spike protein contains the epitope(s) that is capable of inducing PEDV-neutralizing antibodies. In addition, the region that corresponds to the neutralizing epitope of TGEV may also be involved in neutralizing PEDV, although the two viruses are serologically quite distinct. Finally, the amino acid sequences that are deduced from the genes for the determined-neutralizing epitope were highly homologous among the PEDV strains that were isolated from different geographical areas, which suggests conservation of the antigen gene.

Modulation of antigen-specific immune responses by the oral administration of a traditional medicine, bo-yang-hwan-o-tang.

Bo-yang-hwan-o-tang (BHT) has long been used to treat cancer in traditional Korean medicine and is believed to have immune-modulating activity. This study investigated the effect of BHT on the induction of antigen-specific immune responses using hen egg-white lysozyme (HEL) as a model antigen system. Oral administration of BHT enhanced both HEL-specific humoral and lymphocyte proliferative responses in HEL low-responder mice. Feeding BHT to the mice increased INF-gamma levels, but did not change IL-4 levels. Interestingly, however, the oral BHT feeding significantly increased HEL-specific antibodies of the IgG1, IgG2b, and IgG3 subtypes, which are associated with the direct stimulation of B cells. This indicates that BHT treatment enhances anti-HEL-specific humoral immune responses via the direct stimulation of B lymphocytes rather than by selective priming of specific subtypes of the helper T-cell population. This conclusion was supported by in vitro experiments, in which the presence of BHT significantly augmented B-cell mitogen-mediated proliferation of mouse splenocytes. This augmentation was closely associated with a glycoprotein with a molecular weight of around 100 kDa. The results suggest that BHT modulates antigen-specific immune responses, and might be used as a therapeutic agent for patients who need enhanced immune function.