Hong Hee Chang

Immunogenicity and protective effect of recombinant Brucella abortus Ndk (rNdk) against a virulent strain B. abortus 544 infection in BALB/c mice.

In this study, we particularly evaluated the protective effect of recombinant protein encoded by Brucella abortus 544 ndk (nucleoside diphosphate kinase) gene against B. abortus infection in the BALB/c mice. Cloning and expression of B. abortus Ndk was accomplished by PCR amplification into a pMAL expression system, and purification of a recombinant Ndk (rNdk). As for the determination of IgG responses, rNdk induced vigorous IgG production, especially higher in IgG2a compared to IgG1 with titers of 5.2 and 4.8, respectively, whereas titers of these in mice immunized with MBP were 2.4 of IgG2a and 2.6 of IgG1. The analysis of cytokine has revealed that rNdk can strongly induce production of IFN-γ as well as proinflammatory cytokines (TNF, MCP1 and IL-6) but not much IL-10, suggesting rNdk elicited predominantly cell-mediated immune responses. Furthermore, the spleen proliferation and bacterial burden in the spleen of rNdk immunized mice were significantly lower than those of MBP-immunized mice against virulent B. abortus challenge (P < 0.01). Conclusionly, rNdk immunization enables to elicit both of the humoral and cellular response, ultimately enhancing protection level in experimental mice, suggesting that rNdk of B. abortus might be a useful candidate for subunit vaccine for brucellosis in animals.

Anticoccidial effect of supplemental dietary Galla Rhois against infection with Eimeria tenella in chickens

The anticoccidial effects of Galla Rhois (GR) powder, which contains a major tannin-derived component of 52.7%, were evaluated in chickens following oral infection with Eimeria tenella. One-day-old chickens were assigned to five groups (control, unsupplemented, GR 0.5% supplemented [GRS 0.5%], GRS 1.0% [GRS 1.0%] and salinomycin supplemented [SS]). The chickens were fed a standard diet supplemented or not supplemented with GR or salinomycin for 10 days prior to infection. The birds received the supplemented diets continuously until 10 days post infection. The effects of GR on a E. tenella infection were evaluated by several parameters, including body weight gain, feed intake, oocyst excretion, bloody diarrhoea, and lesion scores. Infected chickens on the GRS and SS diets had a relatively moderate body weight loss (reduction ratio < 15%) and improved feed conversion. GRS and SS chickens produced significantly fewer faecal oocysts (P<0.05) and showed milder bloody diarrhoea compared with the E. tenella-infected control group. Furthermore, the lesion scores of both the GRS 0.5% and GRS 1.0% groups were significantly lower than the scores of the unsupplemented group on day 5 post infection. The lesion scores for the GR groups were similar to the scores for the SS group. In conclusion, this study suggests that GR appears to be as efficacious as salinomycin against E. tenella infection. GR supplementation leads to a reduction in infected chickens, although infected chickens are still affected compared with the uninfected control group. GR-based diets may be beneficial in preventing or treating coccidial infections in poultry.

Characterization of betaine aldehyde dehydrogenase (BetB) as an essential virulence factor of Brucella abortus

The pathogenic mechanisms of Brucellosis used to adapt to the harsh intracellular environment of the host cell are not fully understood. The present study investigated the in vitro and in vivo characteristics of B. abortus betaine aldehyde dehydrogenase (BetB) (Gene Bank ID: 006932) using a betB deletion mutant constructed from virulent B. abortus 544. In test under stress conditions, including osmotic- and acid stress-resistance, the betB mutant had a lower osmotic-resistance than B. abortus wild-type. In addition, the betB mutant showed higher internalization rates compared to the wild-type strain; however, it also displayed replication failures in HeLa cells and RAW 264.7 macrophages. During internalization, compared to the wild-type strain, the betB mutant was more adherent to the host surface and showed enhanced phosphorylation of protein kinases, two processes that promote phagocytic activity, in host cells. During intracellular trafficking, colocalization of B. abortus-containing phagosomes with LAMP-1 was elevated in betB mutant-infected cells compared to the wild-type cells. In mice, the betB mutant was predominantly cleared from spleens compared to the wild-type strain after 2 weeks post-infection, and the vaccination test with the live betB mutant showed effective protection against challenge infection with the virulent wild-type strain. These findings suggested that the B. abortus betB gene substantially affects the phagocytic pathway in human phagocytes and in host cells in mice. Furthermore, this study highlights the potential use of the B. abortus betB mutant as a live vaccine for the control of brucellosis.

NDRG2 correlated with favorable recurrence-free survival inhibits metastasis of mouse breast cancer cells via attenuation of active TGF-β production

N-myc downstream-regulated gene 2 (NDRG2) has been studied for its inhibitory effects against growth and metastasis of many tumor cell types. In this study, we showed NDRG2 expression was correlated with favorable recurrence-free survival of patients with breast cancer and inhibited metastasis of breast cancer cells (4T1). NDRG2 expression was examined in 189 breast carcinoma tissues and paired normal breast tissues using immunohistochemistry. Histological and clinicopathological data were correlated using Pearsons chi-square test of independence. NDRG2 expression in human breast cancer tissues was inversely associated with lymph node metastasis and pTNM stage. Furthermore, patients with breast cancer with a high level of NDRG2 expression showed favorable recurrence-free survival (P = 0.038). To study the effect of NDRG2 on metastasis in vivo, we established an NDRG2-overexpressing mouse breast cancer cell line (4T1-NDRG2) and measured the metastasis and survival of 4T1-NDRG2 tumor-bearing mice. To test whether transforming growth factor β (TGF-β)- mediated metastasis of 4T1 was inhibited by NDRG2 expression, TGF-Smad-binding element (SBE)-luciferase activity and/or measurement of active TGF-β were performed in cell or tumor tissue level. 4T1-NDRG2 cells grew gradually and showed less metastatic activity in vivo and low invasiveness in vitro. 4T1-NDRG2 cells showed lower SBE-luciferase activity and lower level of active autocrine TGF-β than 4T1-Mock did. Correctly, our data show that NDRG2 significantly suppress tumor metastasis by attenuating active autocrine TGF-β production, and the attenuation might be typically associated with the favorable recurrence-free survival of patients clinically.

Increase of Available Phosphorus by Fly‐Ash Application in Paddy Soils

Abstract Fly ash from the coal‐burning industry may be a potential inorganic soil amendment to increase rice productivity and to restore the soil nutrient balance in paddy soil. In this study, fly ash was applied at rates of 0, 40, 80, and 120 Mg ha−1 in two paddy soils (silt loam in Yehari and loamy sand in Daegok). During rice cultivation, available phosphorus (P) increased significantly with fly ash application, as there was high content of P (786 mg kg−1) in the applied fly ash. In addition, high content of silicon (Si) and high pH of fly ash contributed to increased available‐P content by ion competition between phosphate and silicate and by neutralization of soil acidity, respectively. With fly‐ash application, water‐soluble P (W‐P) content increased significantly together with increasing aluminum‐bound P (Al‐P) and calcium‐bound P (Ca‐P) fractions. By contrast, iron‐bound P (Fe‐P) decreased significantly because of reduction of iron under the flooded paddy soil during rice cultivation. The present experiment indicated that addition of fly ash had a positive benefit on increasing the P availability.

Intracellular replication inhibitory effects of Galla Rhois ethanol extract for Brucella abortus infection

ETHNOPHARMACOLOGICAL RELEVANCE Galla Rhois (GR) has long been applied in traditional Korean and Oriental medicine. Although GR has an anti-bacterial effect, the anti-bacterial mechanism and therapeutic efficiency of GR for intracellular parasitic Brucella infection are still unclear. AIM OF THE STUDY The objective of this study was to investigate the antibacterial and therapeutic effects of GR ethanol extract (GRE), which is a natural antibacterial component for the treatment of Brucella abortus infection. MATERIALS AND METHODS The antibacterial activity of GRE towards Brucella abortus was evaluated by incubating Brucella abortus with GRE. Following treatment with GRE, Brucella abortus adherence, uptake, intracellular growth, and intracellular trafficking in macrophages were monitored. Mice were infected intraperitoneally with Brucella abortus and treated orally with GRE for 14 days, and then the weight and CFUs from each spleen were monitored. RESULTS The viability of Brucella abortus was markedly decreased in a dose-dependent manner. Moreover, Brucella abortus internalization and intracellular growth within macrophages were reduced in GRE-treated cells. The number of bacteria that adhered to GRE-pretreated cells was significantly lower than that of untreated cells. With regards to intracellular trafficking, treatment with GRE augmented the colocalization of Brucella abortus-containing phagosomes with LAMP-1. GRE-treated mice showed considerably decreased weight and bacterial burdens in the spleen compared to untreated mice. CONCLUSION GRE exhibits antibacterial and protective effects on Brucella abortus in vitro and in vivo. These results highlight the beneficial effects of GRE in the prevention and treatment of brucellosis.

Characterization of culture supernatant proteins from Brucella abortus and its protection effects against murine brucellosis

In this study, we characterized the secreted proteins of Brucella abortus into the enriched media under the bacterial laboratory growth condition and investigated the pathogenic importance of culture supernatant (CS) proteins to B. abortus infection. CS proteins from stationary phase were concentrated and analyzed using 2D electrophoresis. In MALDI TOF/TOF analysis, more than 27 proteins including CuZn SOD, Dps, Tat, OMPs, Adh, LivF, Tuf, SucC, GroEL and DnaK were identified. Cytotoxic effects of CS proteins were found to increase in a dose-dependent manner in RAW 264.7 cells. Upon B. abortus challenge into phagocytes, however, CS proteins pre-treated cells exhibited lower bacterial uptake and intracellular replication compared to untreated cells. Immunization with CS proteins induced a strong humoral and cell mediated immune responses and exhibited significant higher degree of protection against virulence of B. abortus infection compared to mice immunized with Brucella broth protein (BBP). Taken together, these results indicate that B. abortus secreted a number of soluble immunogenic proteins under laboratory culture condition, which can promote antibody production resulted in enhancing host defense against to subsequently bacterial infection. Moreover, further analysis of CS proteins may help to understand the pathogenic mechanism of B. abortus infection and host-pathogen interaction.

Identification of genes contributing to the intracellular replication of Brucella abortus within HeLa and RAW 264.7 cells

Brucella abortus, the causative agent of brucellosis, can survive and replicate within host cells. Understanding bacterial virulence factors and bacteria-host cell interactions is critical for controlling brucellosis, yet very little is known about the virulence strategies and signaling pathways activated in phagocytes during infection to ensure their growth and survival. B. abortus was mutagenized by mini-Tn5Km2 transposon mutagenesis to identify virulence genes related to the internalization and intracellular replication of the bacteria. Of the total 2300 mutants used to infect HeLa cells, 23 mutants defective for intercellular growth and the mutated genes were identified. Sequence analysis of DNA flanking the transposon showed various insertion sites in bacterial genes that might be associated with virulence, including genes associated with lipoproteins, amino acid metabolism, translation, transcription, carbohydrate transport, coenzyme transport, inorganic ion transport, energy metabolism, membrane transport, and cell wall/membrane biogenesis. Moreover, mutants were classified into class I, class II and class III as higher, similar, and lower internalization, respectively, into HeLa cells. Furthermore, defective mutants for intracellular growth in HeLa cells were found to be defective in RAW 264.7 cells. Taken together, we suggest that the identified virulence associated genes might contribute to the intracellular growth and survival of B. abortus in phagocytes.

Redundant effects of ketamine on the pathogenesis and severity of Brucella abortus infection

Brucella abortus is an intracellular bacterium and leading to a serious debilitating disease known as brucellosis. Ketamine is an anesthetic and a sedative that affects the immunomodulatory activities of various immune cells. The current study was to elucidate the role of ketamine in B. abortus infection, focusing on the phagocytic activity and immune response of macrophages. Following incubation of murine macrophages with ketamine, the phagocytosis of B. abortus was markedly reduced compared with the unincubated control. Interestingly, ketamine-incubated cells displayed a decreased intensity of F-actin fluorescence compared with the B. abortus-induced amplification of intensity. Conversely, the intracellular replication of B. abortus within macrophages was notably enhanced by ketamine. Furthermore, the in vivo assessment using a mouse model revealed that continual injections with ketamine led to augmented bacterial burdens in the spleen, which was accompanied by decreased levels of mRNA expression of cytokines in the spleen. The elevations of serum cytokines such as IFN-γ, IL-12 and IL-6, as well as the chemokine MCP-1, were also reduced by ketamine. These findings verify that ketamine suppresses the phagocytic activity and immune response during B. abortus infection. Therefore, the current study might provide novel insights into the potential influences of ketamine on infectious diseases caused by B. abortus, considering the host-pathogen interaction.

Methyl gallate limits infection in mice challenged with Brucella abortus while enhancing the inflammatory response

To investigate the effects of methyl gallate (MG) on murine macrophages, cytokine production and treatment of Brucella abortus infection using a mouse model.