• 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.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.6
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• pp.2847-2851
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• 2012

Purpose: To study the killing effects on osteosarcoma cells of cinobufacini and cisplatin in combination and the related mechanisms so as to explore the chemotherapeutic method with integrated traditional Chinese and Western medicines. Methods: Cinobufacini and cisplatin were applied to OS732 cells singly or jointly and survival rates were measured by MTT assay. Changes in cellular shape were observed with inverted phase contrast and fluorescence microscopy and apoptosis rates were analyzed with flow cytometry (FCM). Immunocytochemistry were used to examine the Fas expression of OS732 cells. Results: The combination of cinobufacini and cisplatin had the effect of up-regulating Fas expression and inducing apoptosis. The survival rate of combined application of 100 ${\mu}g/ml$ cinobufacini and 1 ${\mu}g/ml$ cisplatin on OS-732 cells was significantly lower than with either of the agents alone (p<0.01). Changes in cellular shape and apoptotic rates also indicated the apoptosis-inducing effects of combined application were much enhanced. Conclusion: The combination of cinobufacini and cisplatin demonstrated strong killing effects on OS-732 cells which might be related to up-regulation of Fas expression.

• The Journal of Advanced Prosthodontics
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• v.5 no.1
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• pp.2-8
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• 2013

PURPOSE. Autoclaves and UV sterilizers have been commonly used to prevent cross-infections between dental patients and dental instruments or materials contaminated by saliva and blood. To develop a dental sterilizer which can sterilize most materials, such as metals, rubbers, and plastics, the sterilization effect of an atmospheric pressure non-thermal air plasma device was evaluated. MATERIALS AND METHODS. After inoculating E. coli and B. subtilis the diamond burs and polyvinyl siloxane materials were sterilized by exposing them to the plasma for different lengths of time (30, 60, 90, 120, 180 and, 240 seconds). The diamond burs and polyvinyl siloxane materials were immersed in PBS solutions, cultured on agar plates and quantified by counting the colony forming units. The data were analyzed using one-way ANOVA and significance was assessed by the LSD post hoc test (${\alpha}$=0.05). RESULTS. The device was effective in killing E. coli contained in the plasma device compared with the UV sterilizer. The atmospheric pressure non-thermal air plasma device contributed greatly to the sterilization of diamond burs and polyvinyl siloxane materials inoculated with E. coli and B. subtilis. Diamond burs and polyvinyl siloxane materials inoculated with E. coli was effective after 60 and 90 seconds. The diamond burs and polyvinyl siloxane materials inoculated with B. subtilis was effective after 120 and 180 seconds. CONCLUSION. The atmospheric pressure non-thermal air plasma device was effective in killing both E. coli and B. subtilis, and was more effective in killing E. coli than the UV sterilizer.

• 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.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.27 no.4
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• pp.327-334
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• 1994

This study was undertaken to clarify the effect of killing methods on the morphological and histological changes of plaice, Paralichthys olivaceus muscle at early stage after killing. Killed samples by the three different methods were stored at $5^{\circ}$, and the changes in breaking strength of muscle, morphological observation of myofibrils and histological observation of extracellular spaces through storage were monitored. Samples killed by electrifying in sea water showed the maximum value of breakin strength immediately after killing and then it dropped significantly(p<0.05) until 2.5hrs passed. Breaking strength of samples killed by spiking at the head instantly and dipping in sea water including anesthetic rose steadily over 10hrs and 15hrs after killing, respectively. In myofibrills prepared from dorsal muscles immediately after spiking at the head instantly, A-band, H-band, I-band, and Z-line in sarcomere were clearly distinguishable each other. Due to muscle contraction by electrical stimulation, it was impossible to distinguish H-band from I-band observed in sarcomere immediately after killing for samples killed by electrifying. But, in the cases of samples killed by spiking and dipping, H-band could be observed dimly until 10hrs and 15hrs storage. No extracellular space was observed among muscle cells immediately after spiking at the head instantly. Samples killed by spiking at the head instantly and dipping in sea water including anesthetic showed extracellular spaces among all muscle cells after 15hrs and 25hrs storage, respectively. The other hand, samples killed by electrifying in sea water (110V, 30sec.) showed a few extracellular spaces immediately after killing and then it showed extracellular spaces among all muscle cells after 2.5hrs storage.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.27 no.1
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• pp.41-46
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• 1994

This study was undertaken to clarify the effect of killing methods on physical and rheological changes of plaice, Paralichthys olivaceus muscle at early period after death. Plaices killed by the four different methods(1. spiking at the brain instantly. 2. drowning in air. 3. dipping in 1,000ppm ethylaminobenzoate dissolved sea water as an anesthetic. 4. electrifying in sea water.) were stored at $5^{\circ}C$, and the rigor-index and breaking strength through storage were monitored. The longest onset time of rigor-mortis and full rigor was in the samples killed by dipping in sea water with dissolved anesthetic among all samples, where rigor-mortis began at 20hrs after killing and maximum tension was attained after 56hrs. However, in the cases of plaice electrified in sea water or drowned in air, the onset of rigor-mortis began just after killing and maximum tensions were attained after 9hrs and 13hrs, respectively. The level of breaking strength in the muscle of fish killed by spiking the brain instantly was $950.30{\pm}50.23g$, immediately after killing. The value and time reached around the maximum breaking strength for each of the samples were $1,230.60{\pm}30.32g$ and Ohr (immediately after killing) for samples killed by electrifying in sea water, $1,235.83{\pm}35.37g$ and 2.5hrs for drowning samples, $1,186.29{\pm}55.90g$ and 10hrs for spiking samples, and $1,189.67{\pm}50.32g$ and 15hrs for samples dipped in anesthetic, respectively. From the results above, it could be concluded that electrification in sea water is the most effective method in accelerating rigor-mortis and shortening times of reaching the maximum breaking strength of fresh plaice flesh of all the killing methods at early periods after death, whereas dipping in sea water treated with anesthetic was the most effective way in delaying those changes.

• Biomedical Science Letters
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• v.14 no.4
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• pp.269-274
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• 2008

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common nosocomial pathogens. It is associated with hospitals is now being isolated in the community. The aim of this study was to evaluate the antibacterial effect of photodynamic therapy using Photogem and 630 nm LED on MRSA and methicillin-sensitive Staphylococcus aureus (MSSA). The broth cultured MRSA and MSSA incubated with various concentrations of Photogem (500,50,5 and $0.5{\mu}g/mL$) for 4 h. Then 630 nm LED was given at $9\;J/cm^2$, $20{\mu}l$ of the exposed bacteria solution was inoculated onto agar plate. Plates were incubated for 24 hand colonies were counted. The PDT group was effective in killing MRSA and MSSA at the Photogem dose of $50{\mu}g/mL$. But MSSA is more sensitive than MRSA in photodynamic effect. Other groups (light only, sensitizer only, or no treatment) observed no bacterial cell killing. These results raise the possibility of using PDT with or without antimicrobial drugs to eradicate MRSA and MSSA. In order to confirm this result, we need to further study bacterial death mechanism and in vivo study.

• Tuberculosis and Respiratory Diseases
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• v.44 no.1
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• pp.162-174
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• 1997

Background : Metabolic cooperation via gap junctional intercellular communication (GJIC) is an important mechanism of the bystander effect in gene therapy using the Herpes Simplex Virus thymidine kinase/ganciclovir (HSVtk) "prodrug" system. Since retinoids have been reported to increase GJIC by induction of connexin 43 expression, we hyporthesized that treatment of tumor cells with retinoic acid could augment the bystander effect of the HSVtk/GCV system and result in improved tumor cell killing by enhancing GJIC. Methods : We transferred HSVtk gene to SKHep-J cell line that does not express connexin43, and also transferred the gene to human and murine mesothelioma cell lines that express connexin43. We verified that retinoic acid enhanced GJIC utilizing a functional double-dye transfer study and evaluated the effects of retinoic acid on the growth rate of tumor cells. We then tested the effects of retinoic acid on bystander-mediated cell killing. Results : Addition of all-trans retinoic acid (RA) increased GJIC in cell lines expressing connexin 43 and was asspciated with more efficient in vitro bystander killing in cells transduced with HSVtk via adenoviral and retroviral vectors. In contrast, there was no increase in the efficiency of the bystander effect after exposure to RA in a cell line which had no delectable connexin 43. Conclusion : These results provide evidence that retinoids can augment the efficiency of cell killing with the HSVtk/GCV system by enhancing bystander effect and may thus be a promising new approach to improve responses in gene therapy utilizing the HSVtk system to treat tumors.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.30 no.4
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• pp.589-594
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• 1997

This study was performed to clarify the effect of anesthesia killing and non-bleeding on the physicochemical and rheological properties of plaice, Paralichthys olivaceus muscle at early period after death. Live plaice was killed by the two different methods: spiking at the brain instantly with bleeding and dipping In seawater containing anesthetic (2,000 ppm ethyl-aminobenzoate) for 10 min without bleeding. These samples were stored at $0^{\circ}C$ and used in checking rigor-mortis, ATP breakdown, the content of ATP and its related compounds, breaking strength, and lactate accumulation through storage. The rigor-mortis, ATP breakdown, and lactate accumulation was faster in samples killed by spiking than in samples killed by anesthesia. ATP in samples killed by anesthetic showed little breakdown until 22.5 hrs, but it was decomposed completely after 30 hrs storage. Breaking strength of samples killed by spiking at the brain instantly with bleeding decreased steadily and showed the maximum value over 10 hrs $(2207.3{\pm}60.2g)$. However, in case of the dipping fresh flesh without bleeding in seawater containing anesthetic, the value and time reached around the maximum breaking strength were $2147.8{\pm}29.0g$ and 13 hrs respectively, but it maintained constantly until 20 hrs passed. From these results, it could be suggested that anesthesia killing and non-bleeding is more effective in maintaining firmness of fresh plaice muscle than spiking killing with bleeding at the early period after death.

• YAKHAK HOEJI
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• v.55 no.1
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• pp.39-44
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• 2011

Many reports indicate that $18{\beta}$-glycyrrhetinic acid ($18{\beta}$-GA) from Glycyrrhizae Radix has anti-inflammatory and immunoregulatory activities, whereas reports regarding anticancer activity of the compound are few. In present study, we investigated antitumor effect of $18{\beta}$-GA on tumor caused by A549 cancer cell in mice. Data resulting from the cytotoxicity assay showed that $18{\beta}$-GA caused killing of A549 cells. $LD_{50}$ values of $18{\beta}$-GA were app. 180 ${\mu}M$ and 80 ${\mu}M$, corresponding to 48 hr- and 72 hr-treatments, displaying that the killing activity was more effective as the $18{\beta}$-GA treatment was prolonged. Based on these data, antitumor effect of $18{\beta}$-GA was tested in nude mice. For induction of the tumor, A549 ($3{\times}10^6$ cells/mouse) was injected subcutaneously into the lateral abdomen of nude mice (Balb/c nu/nu). To determine the antitumor effect, nude mice with tumor were given $18{\beta}$-GA (1 mg/200 ${\mu}l$/mouse) intraperitoneally every three days for four times. Tumor-sizes were measured with a caliper for a period of 24 days. Results showed that the $18{\beta}$-GA treatment reduced the tumor-sizes (P<0.05) as compared with negative control nude mice that received diluent (DPBS). The reduction degree was greater than reduction degree by doxorubicin (60 ${\mu}g$/mouse), and the pattern of reduction was almost sustained during the entire period of the observation. In conclusion, our studies demonstrate that $18{\beta}$-GA has antitumor activity to the A549 cancer cell-caused tumor.