• Journal of Microbiology and Biotechnology
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• v.25 no.5
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• pp.738-744
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• 2015

Tuberculosis is one of the most threatening infectious diseases to public health all over the world, for which Mycobacterium tuberculosis (MTB) is the etiological agent of pathogenesis. Ursolic acid (UA) has immunomodulatory function and exhibits antimycobacterial activity. However, the intracellular killing effect of UA has yet to be elucidated. The aim of this study was to evaluate the intracellular killing effect of UA during mycobacterial infection. The intracellular killing activity of UA was evaluated in the macrophage cell line THP-1 by the MGIT 960 system as well as by CFU count. The production of reactive oxygen species (ROS) and the level of nitric oxide (NO) were measured using DCF-DA and Griess reagent, respectively. Phagocytosis was observed by a fluorescence-based staining method, and the colony forming units were enumerated on 7H11 agar medium following infection. In addition, MRP8 mRNA expression was measured by qRT-PCR. UA significantly decreased the number of intracellular Mycobacterium through generation of ROS and NO. In addition, it profoundly activated the phagocytosis process of THP-1 cells during MTB-infection. Furthermore, our data demonstrated that UA activated the phagocytosis process in human monocyte cells through MRP8 induction. These data suggest that UA firmly contributes to the intracellular killing effect of macrophages during mycobacterial infection.

• Tuberculosis and Respiratory Diseases
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• v.53 no.1
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• pp.5-16
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• 2002

Background : Though mononuclear phagocytes serve as the final effectors in killing intracellular Mycobacterium tuberculosis, the bacilli readily survive in the intracellular environment of resting cells. The mechanisms through which cellular activation results in the intracellular killing is unclear. In this study, we sought to explore an in vitro model of a low-level infection of human mononuclear phagocytes with MAC and $H_{37}Ra$ and determine the extent of the lymphocyte dependent cytotoxicity of human monocytes and alveolar macrophages. Materials and Methods : The peripheral monocytes were prepared using the Ficoll gradient method from PPD positive healthy people and tuberculosis patients. The alveolar macrophages were prepared from PPD positive healthy people via a bronchoalveolar lavage. The human mononuclear phagocytes were infected at a low infection rate (bacilli:phagocyte 1:10) with MAC(Mycobacterium avium) and Mycobacterium tuberculosis $H_{37}Ra$. Non-adherent cells(lymphocyte) were added at a 10:1 ratio. After 1,4, and 7 days culture in $37^{\circ}C$, 5% CO2 incubator, the cells were harvested and inoculated in a 7H10/OADC agar plate for the CFU assay. The bacilli were calculated with the CFU/$1{\times}10^6$ of the cells and the cytotoxicity was expressed as the log killing ratio. Results : The intracellular killing of MAC and $H_{37}Ra$ within the monocyte was greater in patients with tuberculosis compared to the PPD positive controls (p<0.05). Intracellular killing of MAC and $H_{37}Ra$ within the alveolar macrophage appeared to be greater than that within the monocytes of the PPD positive controls. There was significant lymphocyte dependent inhibition of intracellular growth of the mycobacteria within the monocytes in both the controls and tuberculosis patients and within the macrophages in the controls(p<0.05). There was no specific difference in the virulence between the MAC and the $H_{37}Ra$. Conclusion : This study is an in vitro model of a low-level infection with MAC and $H_{37}Ra$ of human mononuclear phagocytes. The intracellular cytotoxicity of the mycobacteria within the phagocytic cells was significantly lymphocyte dependent. During the 7 days culture after the intracellular phagocytosis, the actual confinement of the mycobacteria was observed within the monocytes of tuberculosis patients and the alveolar macrophages of the controls as in the case of adding lymphocytes.

• Journal of Veterinary Science
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• v.23 no.3
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• pp.48.1-48.12
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• 2022

Background: The poor intracellular concentration of enrofloxacin might lead to treatment failure of cow mastitis caused by Staphylococcus aureus small colony variants (SASCVs). Objectives: In this study, enrofloxacin composite nanogels were developed to increase the intracellular therapeutic drug concentrations and enhance the efficacy of enrofloxacin against cow mastitis caused by intracellular SASCVs. Methods: Enrofloxacin composite nanogels were formulated by an electrostatic interaction between gelatin (positive charge) and sodium alginate (SA; negative charge) with the help of CaCl₂ (ionic crosslinkers) and optimized by a single factor test using the particle diameter, zeta potential (ZP), polydispersity index (PDI), loading capacity (LC), and encapsulation efficiency (EE) as indexes. The formation mechanism, structural characteristics, bioadhesion ability, cellular uptake, and the antibacterial activity of the enrofloxacin composite nanogels against intracellular SASCVs strain were studied systematically. Results: The optimized formulation was comprised of 10 mg/mL (gelatin), 5 mg/mL (SA), and 0.25 mg/mL (CaCl₂). The size, LC, EE, PDI, and ZP of the optimized enrofloxacin composite nanogels were 323.2 ± 4.3 nm, 15.4% ± 0.2%, 69.6% ± 1.3%, 0.11 ± 0.02, and -34.4 ± 0.8 mV, respectively. Transmission electron microscopy showed that the enrofloxacin composite nanogels were spherical with a smooth surface and good particle size distributions. In addition, the enrofloxacin composite nanogels could enhance the bioadhesion capacity of enrofloxacin for the SASCVs strain by adhesive studies. The minimum inhibitory concentration, minimum bactericidal concentration, minimum biofilm inhibitory concentration, and minimum biofilm eradication concentration were 2, 4, 4, and 8 ㎍/mL, respectively. The killing rate curve had a concentration-dependent bactericidal effect as increasing drug concentrations induced swifter and more radical killing effects. Conclusions: This study provides a good tendency for developing enrofloxacin composite nanogels for treating cow mastitis caused by intracellular SASCVs and other intracellular bacterial infections.

• Biomolecules & Therapeutics
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• v.11 no.3
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• pp.174-177
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• 2003

The present experiment was performed to investigate the protective effects of paeonol isolated from Moutan Cortex Radicis on primary cultured rat hepatocytes exposed to Br-A23187 ($Ca^{2+}$ ionophore). Br-A23187 is frequently used as a model of cell killing as inducing both necrotic and apoptotic cell death. Hepatocytes were isolated by collagenase perfusion from livers of fasted male Sprague Dawley rats and cultured overnight. Cell viability was determined by propidium iodide using fluorocytometry in Krebs-Ringer-HEPES buffer at pH 7.4. In addition, intracellular calcium was measured by excitation at 340 and 380 nm and emission at 505 nm using a luminescence spectrophotometer. Paeonol (20-100 ${\mu}M$) inhibited cell killing induced by 10 ${\mu}M$ Br-A23187, in a dose-dependent manner. Paeonol also reduced increased intracellular calcium level when hepatocytes were exposed to Br-A23187. Therefore, the present results suggest that paeonol protects the hepatocytotoxicity induced by Br-A23187, via inhibiting the influx of calcium into into rat hepatocytes.

• Journal of Microbiology and Biotechnology
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• v.25 no.6
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• pp.946-950
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• 2015

Recently, it has become a struggle to treat tuberculosis with the current commercial antituberculosis drugs because of the increasing emergence of multidrug-resistant (MDR) tuberculosis and extensively drug-resistant (XDR) tuberculosis. We evaluated here the antimycobacterial activity of tamoxifen, known as a synthetic anti-estrogen, against eight drugsensitive or resistant strains of Mycobacterium tuberculosis (TB), and the active intracellular killing of tamoxifen on TB in macrophages. The results showed that tamoxifen had antituberculosis activity against drug-sensitive strains (MIC, 3.125-6.25 µg/ml) as well as drugresistant strains (MIC, 6.25 to 12.5 µg/ml). In addition, tamoxifen profoundly decreased the number of intracellular TB in macrophages in a dose-dependent manner.

• The Journal of Korean Obstetrics and Gynecology
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• v.25 no.3
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• pp.27-39
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• 2012

Objectives: Recently Ciprofloxacin, used in the treatment of mastitis, showed many serious side effects. The object of this study was to recognize whether TNS and KWT can be used in the treatment of mastitis by observing the in vitro antibacterial effects of TNS and KWT aqueous herbal extracts against S. aureus. Methods: Antibacterial activities of TNS and KWT aqueous extracts against S. aureus ATCC 25923 were detected using standard agar microdilution methods. In addition, the effects on the bacterial growth curve were monitored at MIC and $MIC{\times}2$ levels. The effects on the intracellular killing and bacterial invasion of individual test materials were also observed using Raw 264.6 and MCF-7. The results were compared with Ciprofloxacin, a second generation of quinolone antibiotics in the present study. Results: MIC of aqueous extracts of TNS and KWT against S. aureus were detected as ($0.313{\pm}0.107$) and ($0.137{\pm}0.053$) mg/ml, respectively. MIC of Ciprofloxacin was detected as ($0.469{\pm}0.297$) ${\mu}g/ml$ at same conditions. In addition, TNS, KWT aqueous herbal extracts and Ciprofloxacin were also showed marked dosage-dependent inhibition of bacterial growth, and dramatical inhibitions on the both intracellular killing assays and bacterial invasion using Raw 264.6 and MCF-7 cells were detected. Conclusions: The results obtained in this study suggest that TNS and KWT aqueous herbal extracts showed antibacterial effects against S. aureus, and they also showed dosage-dependent inhibitory effects on the bacterial growth. And they showed the significant intracellular killing and bacterial invasion effects. It means, KWT and TNS may show more potent anti-infectious effects against S. aureus in vivo.

• The Journal of Korean Obstetrics and Gynecology
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• v.26 no.1
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• pp.25-40
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• 2013

Objectives: The object of this study was to observe the in vitro antibacterial effects of five single(Pulsatillae Radix, Patrinae Radix, Sanguisorbae Radix, Sophorae Flos, and Sophorae Radix) aqueous herbal extracts, traditionally used for treating various gynecological diseases including mastitis in Korea, against Staphylococcus aureus. Methods: Antibacterial activities against Staphylococcus aureus of aqueous extracts of Pulsatillae Radix, PatrinaeRadix, Sanguisorbae Radix, Sophorae Flos, and Sophorae Radix were detected using standard agar microdilution methods. In addition, the effects on the bacterial growth curve were also monitored at Minimal Incubation Concentration(MIC) and $MIC{\times}2$ levels. The effects on the intracellular killing and bacterial invasion of individual test materials were also observed using murine macrophage(Raw 264.7) and human mammary gland carcinoma cell(MCF-7). Results: MIC of aqueous extracts of Pulsatillae Radix, Patrinae Radix, Sanguisorbae Radix, Sophorae Flos, and Sophorae Radix against Staphylococcus aureus were detected as $0.215{\pm}0.107$ mg/ml, $0.273{\pm}0.107$ mg/ml, $0.469{\pm}0.297$ mg/ml, $11.850{\pm}8.406$ mg/ml, and $0.664{\pm}0.546$ mg/ml, respectively. MIC of Ciprofloxacin was detected as $0.469{\pm}0.297{\mu}g/ml$ at same conditions. In addition, all five single aqueous herbal extracts were also showed marked dosage-dependent inhibition of bacterial growth. The effects of intracellular killing with Raw 264.7 and inhibition of bacterial invasion with MCF-7 cells were detected, in the order of Sophorae Flos, Pulsatillae Radix, Patrinae Radix, Sanguisorbae Radix and Sophorae Radix aqueous extracts in the present study. Conclusions: The results obtained in this study suggest that all five single aqueous herbal extracts showed antibacterial effects against Staphylococcus aureus and they also showed dosage-dependent inhibitory effects on the bacterial growth. They showed the significant intracellular killing and inhibition of bacterial invasion effects. It means, all five single aqueous herbal extracts may show potent anti-infectious effects against Staphylococcus aureus for mastitis.

• The Journal of Korean Obstetrics and Gynecology
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• v.27 no.1
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• pp.65-80
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• 2014

Objectives: The object of this study was to observe the in vitro antibacterial effects of Gagam-seopyoungjeon aqueous extracts (GGSYJ) against Gardnerella vaginalis and the possible synergic combination effects with clindamycin. Methods: Antibacterial activities against Gardnerella vaginalis of GGSYJ were detected using minimal inhibition concentration (MIC), and the effects on the bacterial growth curve were also monitored at MIC and MIC${\times}$2 levels. The combination effects of GGSYJ with clindamycin were observed by checkboard microtiter assay, and the effects of bacterial growth curve treated with GGSYJ MIC+clindamycin MIC, 1/2 MIC and 1/4 MIC, respectively. The effects on the bacterial invasion and intracellular killing of GGSYJ were also observed using human vaginal epithelial (VK2) and murine macrophage (Raw264.7) cells with combination effects with clindamycin after treatment of GGSYJ MIC+clindamycin 1/2 MIC, 1/4 MIC and 1/6 MIC, respectively. Results: The MIC of clindamycin and GGSYJ against Gardnerella vaginalis were detected as $0.012{\pm}0.006$ (0.004~0.016)${\mu}g/ml$ and $1.016{\pm}0.524$ (0.391~1.563) mg/ml, respectively. Clindamycin and GGSYJ were also showed marked dosage-dependent inhibition of bacterial growth, and significant decreases of viable cells were detected in clindamycin MIC+GGSYJ MIC and clindamycin 1/2 MIC+GGSYJ MIC treatment as compared with each of single clindamycin MIC and GGSYJ MIC treatments. And significant decreases of intraepithelial and intra-macrophage viable bacteria numbers were detected in clindamycin 1/2 MIC+GGSYJ 1/2 MIC and clindamycin 1/4 MIC+GGSYJ 1/2 MIC treatment as compared with each of single clindamycin GGSYJ 1/2 MIC treatments, respectively. Conclusions: GGSYJ showed slight antibacterial effects against Gardnerella vaginalis, but they showed dosage-dependent inhibitory effects on the bacterial growth and VK2 epithelial invasions of bacteria with favorable accelerating effects of intracellular killing activities of macrophages. In addition, combination of GGSYJ also increased the inhibitory effects of clindamycin on the epithelial invasions of Gardnerella vaginalis and intracellular killing activities of macrophages against Gardnerella vaginalis as 2-fold higher as compared with clindamycin single treatment, respectively. Therefore, we expected that the clinical dosages of clindamycin can be reduced as 1/2 levels as combination with GGSYJ.

• Tuberculosis and Respiratory Diseases
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• v.53 no.5
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• pp.497-509
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• 2002

Background : The mechanisms through which cellular activation results in intracellular mycobacterial killing is only partially understood. However, in vitro studies of human immunity to Mycobacterium tuberculosis have been largely modeled on the work reported by Crowle, which is complicated by several factors. The whole blood culture is simple and allows the simultaneous analysis of the relationship between bacterial killing and the effect of effector cells and humoral factors. In this study, we attempted to determine the extent to which M. tuberculosis is killed in a human whole blood culture and to explore the role of the host and microbial factor in this process. Methods : The PPD positive subject were compared to the umbilical cord blood and patients with tuberculosis, diabetes and lung cancer. The culture is performed using heparinized whole blood diluted with a culture medium and infected with a low number of M. avium or M. tuberculosis $H_{37}Ra$ for 4 days by rotating the culture in a $37^{\circ}C$, 5% $CO_2$ incubator. In some experiments, methlprednisolone- or pentoxifyline were used to inhibit the immune response. To assess the role of the T-cell subsets, CD4+, CD8+ T-cells or both were removed from the blood using magnetic beads. The ${\Delta}$ log killing ratio was defined using a CFU assay as the difference in the log number of viable organisms in the completed culture compared to the inoculum. Results : 1. A trend was noted toward the improved killing of mycobacteria in PPD+ subjects comparing to the umbilical cord blood but there was no specific difference in the patients with tuberculosis, diabetes and lung cancer. 2. Methylprednisolone and pentoxifyline adversely affected the killing in the PPD+ subjects umbilical cord blood and patients with tuberculosis. 3. The deletion of CD4+ or CD8+ T-lymphocytes adversely affected the killing of M. avium and M. tuberculosis $H_{37}Ra$ by PPD+ subjects. Deletion of both cell types had an additive effect, particularly in M. tuberculosis $H_{37}Ra$. 4. A significantly improved mycobacterial killing was noted after chemotherapy in patients with tuberculosis and the ${\Delta}$ logKR continuously decreased in a 3 and 4 days of whole blood culture. Conclusion : The in vitro bactericidal assay by human whole blood culture model was settled using a CFU assay. However, the host immunity to M. tuberculosis was not apparent in the human whole blood culture bactericidal assay, and patients with tuberculosis showed markedly improved bacterial killing after anti-tuberculous chemotherapy compared to before. The simplicity of a whole blood culture facilitates its inclusion in a clinical trial and it may have a potential role as a surrogate marker in a TB vaccine trial.

• Molecules and Cells
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• v.21 no.1
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• pp.104-111
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• 2006

Gene therapy with nonviral vectors using the suicide gene/prodrug activating system of herpes simplex virus type-1 thymidine kinase (HSV1-TK)/ganciclovir (GCV) is inefficient in killing malignant tumor cells due to two major factors: (a) an unsatisfactory bystander effect; (b) short-lived expression of the protein. To study the capacity of the protein transduction domain (PTD) of HIV-1 TAT protein to enhance HSV1-TK/GCV cancer gene therapy, we constructed three fusion proteins TAT-TK, TK-TAT and TK. TAT-TK retained as much enzyme activity as TK, whereas that of TK-TAT was much lower. TAT-TK can enter HepG2 cells and much of it is translocated to the nucleus. The transduced HepG2 cells are killed by exogenously added GCV and have bystander effects on untransduced HepG2 cells. Most importantly, the introduced recombinant protein is stable and remains functional for several days at least, probably because nuclear localization protects it from the cytoplasmic degradation machinery and provides access to the nuclear transcription machinery. Our results indicate that TAT fusion proteins traffic intercellularly and have enhanced stability and prodrug cell killing activity. We conclude that TAT has potential for enhancing enzyme prodrug treatment of liver cancers.