All-trans retinoic acid (ATRA) is a derivative of vitamin A and exhibits anticancer activity against acute promyelocytic leukemia. Fenbendazole (FBZ) is a benzimidazole anthelmintic that has wide safety margin and low toxicity. Recently, FBZ has been found to have anticancer activity by destabilizing microtubules. In this study, we treat ATRA and FBZ on HL-60 cells, a human leukemia cell line, to investigate the synergistic effects of two drugs, and the potential anticancer mechanism. ATRA and FBZ significantly decreased the metabolic activity of HL-60 cells at 0.04 μM ATRA. Cell viability of ATRA-treated HL-60 cells decreased in a concentration-dependent manner and more decreased by FBZ. N-acetyl cysteine, an inhibitor of reactive oxygen species production, significantly increased the metabolic activity of the cells treated with ATRA and FBZ. Hoechst 33342 and propidium iodide staining showed the presence of broken nuclei in the HL-60 cells treated with ATRA and FBZ. And also, an apoptosis analysis demonstrated that 0.2 μM FBZ increased the percentages of cells in apoptosis and necrosis. In contrast, 0.04 μM ATRA showed no significant difference. Based on multiple assays, ATRA and FBZ showed not synergistic, but additive effect on HL-60 cells. This study may provide researchers and clinicians in cancer-related fields with some valuable information regarding the application of ATRA and FBZ.

Fenbendazole (FBZ) is one of the safest anthelmintic drugs. FBZ has been found to have anti-cancer effects by destabilizing microtubules. In this study, a synergistic effect of paclitaxel (PA), a microtubule-stabilizing anti-cancer agent, and FBZ was investigated on HL-60 cells, a human leukemia cell line. The metabolic activity of cells significantly decreased and the nucleus morphology upon the treatment of FBZ and PA based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay and Hoechst 33342 staining, respectively. To investigate the involvement of reactive oxygen species (ROS), the metabolic activity of the cells after treatment of N-acetyl-L-cysteine (NAC) was measured. Indeed, NAC significantly increased the metabolic activity of the cells treated with FBZ and PA, suggesting that both drugs affect at least in part via ROS. Furthermore, FBZ and PA increased cell death in an annexin V- fluorescein isothiocyanate/propidium iodide staining assay. Taken together, FBZ and PA have a synergistic anti-cancer activity on HL-60 cells at a certain concentration. These results may provide researchers and clinicians in cancer-related fields with some valuable information to broaden the use of FBZ.

Biosilica is a material extracted from the shell (cell wall) of Melosira nummuloides, a type of sea diatom, and is one of the widely distributed biominerals. Recently, some studies have revealed that biosilica has a characteristic bio-modulatory activity compared to synthetic silica. However, there has been little research on the effects and action mechanisms of biosilica in immune cells. In this study, we investigated the effect of biosilica water on mouse dendritic cells (DCs), the most potent antigen-presenting cells in immunity, and whether it may alter the function of DCs. Biosilica water decreased the metabolic activity of DCs at 20% concentration (v/v) and the production of IL-1 beta in a concentration-dependent manner. And also, more cells with fragmented nuclei were observed in the DCs treated with 20% biosilica water compared to other treatment groups. The mixed leukocyte response experiment showed the biosilica water-treated DCs significantly modulated the metabolic activity and proliferation of allogeneic spleen cells compared to control DCs. This result suggests that biosilica water may modulate the antigen-presenting capability of DCs. Taken together, this study shows the immunomodulatory activity of biosilica on DCs and may affect immune responses. Further research is needed to investigate the immunological activity of biosilica water.

Enrofloxacin (EFX) is one of the 2nd generation quinolone antibiotics and is now widely used as a broad-spectrum antibiotic for industrial animals. Previous study showed that EFX reduces the cellular metabolic activity of spleen cells and modulates the inflammatory responses. However, little is known about its toxicity on bone marrow (BM) cells. In this study, BM cells were treated with EFX and cellular metabolic activity, cell death, the change of neutrophil (CD11b+Gr1+ cells) proportion, and antigen uptake ability of granulocytes were measured. Compared to the control, EFX-treated cells showed the decrease of cellular metabolic activity, the increase of cell death, and the decreased proportion of neutrophils. In contrast, the antigen uptake ability of granulocytes in BM cells was increased by EFX. These data suggest that EFX has only limited toxicity on BM cells. And also, EFX is safe on BM cells in a range of concentration, 6.25 – 25 μg/mL. This study can provide available data for the safety or toxicity of EFX.

American foulbrood (AFB) is caused by the bacterium Paenibacillus larvae, which is highly contagious and often lethal to honeybee broods. To control AFB, rapid diagnostic tools including those based on immunological methods are required. We produced several specific mouse monoclonal antibodies (MAbs) against P. larvae. Interestingly, a few of the MAbs were revealed to be an IgM-type antibody. To ascertain the effects of adjuvants on immunoglobulin isotype switching, BALB/c mice were immunized with various adjuvants, i.e., Freund's adjuvant (FA), Alum adjuvant, and AddaVax™ followed by the generation of hybridoma that secreted monoclonal antibodies to P. larvae. In the case of AddaVax™, all screened hybridoma clones secreted IgG-type MAbs, whereas hybridomas generated by Alum and FA secreted 91.25% (7/80) and 66.67% (11/33) respectively, IgG-type MAbs. Although the mechanism of incomplete immunoglobulin isotype switching associated with the P. larvae antigen needs further study, our results indicate that the applied adjuvants can have a significant effect on immunoglobulin isotype switching results.

Disulfiram is a drug used to treat alcohol dependence. Recent studies have shown that disulfiram also has anti-cancer effects. Considering that many anti-cancer agents have side effects, including immunosuppression, it is important to check if disulfiram has some cytotoxicity to immune cells. In this study, mouse spleen cells were treated with disulfiram and the metabolic activity was measured. Disulfiram increased the cell death of spleen cells according to annexin V-FITC/PI staining analysis. In addition, disulfiram decreased the mitochondrial membrane potential of spleen cells. The toxicity of disulfiram was concentration dependent. Interestingly, disulfiram affected the population of lymphocytes and the subset of spleen cells was altered. This study provides clinicians and researchers with valuable information regarding the toxicity of disulfiram to mouse spleen cells, particularly lymphocytes.

Oriental medicine uses many herbs with biological activity. Among these, some have anti-inflammatory activities, but their action mechanisms have yet to be fully elucidated. Therefore, in this study, we harvested mouse bone marrow cells (BMs) and treated them with lipopolysaccharide (LPS), after which they were treated with the extracts of four medicinal herbs. The metabolic activity, cell death ratio, and tumor necrosis factor (TNF)-alpha production of the BMs was then evaluated. Among the four medicinal herbs, Caesalpinia sappan (CS) significantly decreased the metabolic activity, mitochondrial membrane potential of BMs, and production of TNF-alpha, especially in the presence of LPS. Moreover, CS increased the cell death of BMs stained with propidium iodide. Taken together, these findings indicate that CS inhibited the biological activities of LPS-treated BMs in multiple assays, confirming that CS has anti-inflammatory activity.

Doxorubicin has been used to treating cancers, including breast cancer, bladder cancer, and acute lymphocytic leukemia, however, few studies have investigated its anti-inflammatory activity. In this study, we used mouse spleen cells treated with lipopolysaccharide (LPS), a representative inflammatory agent to investigate the effects of doxorubicin. Specially, we investigated the effects of doxorubicin on metabolic activity, cell size, cell death, and cytokine production of LPS-treated spleen cells. Doxorubicin significantly decreased the metabolic activity, even when applied at relatively low concentrations (1.6-8 ng/mL). To investigate the potential mechanism, we measured the mitochondrial membrane potential (MMP) of the LPS-treated spleen cells using Rhodamine 123. Doxorubicin decreased MMP and cell size, and induced cell death. Furthermore, doxorubicin suppressed the production of tumor necrosis factor (TNF)-alpha, a representative cytokine, in LPS-treated spleen cells. Taken together, doxorubicin decreased metabolic activity and the production of inflammatory cytokines, while increasing the death of LPS-induced hyperactivated spleen cells. This results will enable broader application of doxorubicin, as an anti-inflammatory agent, in clinical and research fields.

Many kinds of medicinal herbs have been used to treat inflammation in Oriental medicine. However, there few studies have investigated the anti-inflammatory activity of medicinal herbs. In this study, we used mouse bone marrow cells (BMs) treated with lipopolysaccharide (LPS), a simulator of osteomyelitis, to screen medicinal herbs having anti-inflammatory activity. Specifically, we investigated the activity of an extract of Rhus chinensis (RC) using metabolic activity and cytokine production of the BMs treated with LPS and RC. The metabolic activity of BMs was measured using Cell Counting Kit-8® solution. RC decreased the metabolic activity of LPS-treated BMs. A viability assay using trypan blue solution demonstrated that RC marginally decreased the viability of LPS-treated BMs. Flow cytometry analysis revealed that RC decreased the mitochondrial membrane potential of BMs, regardless of LPS treatment. To investigate the anti-inflammatory activity of RC, we measured the production of tumor necrosis factor (TNF)-alpha and interleukin (IL)-10 in BMs. LPS increased the production of both cytokines in BMs. Interestingly, RC induced a greater increase in IL-10 than TNF-alpha in LPS-treated BMs. Taken together, RC decreased metabolic activity and modulated the production of inflammation-related cytokines in LPS-treated BMs. These findings suggest that RC can be used as a medicinal herb with anti-inflammatory activity.

Fucoidan is a sulfated polysaccharide that is purified from brown algae, such as Fucus vesiculosus. This compound has multiple biological activities including immune-stimulating, and anti-viral activities. We recently demonstrated that the cytotoxicity of fucoidan can be dependent on the batch of its production and its molecular weight. In a previous study, fucoidan B exerted cytotoxicity toward mouse spleen cells. To confirm the biological activity of Fucoidan B, we cultured HL-60 cells, a human leukemia cell line, and treated then with fucoidan. The metabolic activity of the HL-60 cells decreased in response to treatment by fucoidan. Moreover, the morphology of HL-60 treated by fucoidan changed. To investigate the fucoidan’s effects, we analyzed the size and level of Annexin V/propidium iodide staining of HL-60 cells using a flow cytometer. Fucoidan consistently induced cell death, including apoptosis of HL-60 cells. As potential mechanisms, fucoidan destabilized the mitochondrial membrane potential and altered the production of reactive oxygen species in HL-60 cells. Taken together, these results suggest that fucoidan has anti-tumor activity on HL-60 cells via destabilization of mitochondrial membrane potential. The present study demonstrates that fucoidan can be used as an anti-cancer agent for leukemia.

Fucoidan is a sulfated polysaccharide, purified from brown algae. It has multiple biological activities including anti-cancer and anti-inflammatory effects. Our previous reports demonstrated that fucoidan can stimulate spleen cells and especially high molecular weight fucoidan is responsible for the immunostimulatory activity. However, we recently found that the activity of fucoidan can be dependent on its individual batch or sources. Four different fucoidans (fucoidan A, fucoidan B, high molecular weight fucoidan, and low molecular weight fucoidan) were used for this study. MTT assay and flow cytometry analysis were performed for analysis of the activity of fucoidan. MTT assay showed that fucoidan B significantly decreased the cellular activity of spleen cells compared to fucoidan A. In addition, fucoidan B consistently killed spleen cells based on the cell size by flow cytometry analysis and the morphology by an inverted microscope. To elucidate the detailed mechanisms of cytotoxicity, fucoidan B-treated spleen cells were stained with Rhodamine 123 solution and Annexin V-FITC/propidium iodide for measurement of mitochondrial membrane potential (MMP) and early/late apoptosis, respectively. From these two assays, fucoidan B decreased the MMP and induced early apoptosis of spleen cells. Taken together, we suggest that different batches or origin of fucoidan may have differential activities on spleen cells, immunostimulatory and cytotoxic activity. The present study may provide some valuable information regarding use of fucoidan in the clinical area and in basic research.

Taxol is an anti-cancer agent that stabilizes the microtubules of cancer cells, resulting in inhibition of mitosis and thus preventing the proliferation of cancer cells. However, many anti-cancer agents including taxol work on normal cells as well as cancer cells, resulting in side effects such as immunosuppression. A marine algae-derived sulfated polysaccharide, fucoidan, an anti-cancer agent, also showed immunostimulating effects. This study investigated the effects of fucoidan on taxol-treated spleen cells. Spleen cells were treated with taxol in a concentration-dependent manner and in combination with fucoidan. MTT assay and flow cytometry analysis were performed to measure the viability and activity of treated cells. Two assays demonstrated that taxol induced the death of spleen cells. Fucoidan clearly inhibited the cell death induced by taxol. In addition, fucoidan enhanced the production of nitric oxide in spleen cells, which was decreased by taxol. Taken together, taxol can induce the cell death of spleen cells, a major type of immune cells and fucoidan protects spleen cells from taxol-induced cell death. This finding suggests that taxol and fucoidan can be used in combination for lowering the immunosuppressive effects of taxol.

In photobiophysics, biophoton means a kind of biological energy which enhances most metabolisms of the body. To investigate the immuno-enhancing effects of biophoton energy projector (BEP) producing light energy, pigs were irradiated with BEP for 8 weeks. Swine peripheral blood mononuclear cells (sPBMCs) were isolated from the blood of irradiated pigs. In this study, the antigen uptake and mitochondrial membrane potential of sPBMCs were measured by flow cytometric analysis. The irradiation of BEP increased the antigen uptake of sPBMCs. For functional analysis, the production of Bordetella bronchiseptica-specific IgG, measured using antigen-specific ELISA, was increased during the period of BEP irradiation. Taken together, the results suggest that the irradiation of BEP has immune-enhancing effects on sPBMCs.

Mycoplasma hyopneumoniae (M. hyopneumoniae) is one of the causative bacteria that can induce chronic enzootic pneumonia, resulting in low production in the swine industry. Potentiation of porcine reproductive and respiratory syndrome virus-induced pneumonia by M. hyopneumoniae has also been recognized. Although some available vaccines have been developed for prevention of M. hyopneumoniae infection, protective immunity is still poor. In this study, in order to provide valuable information on vaccine antigen, we investigated the immunogenicity of M. hyopneumoniae on mouse spleen cells. Concanavalin A (ConA) and lipopolysaccharide (LPS) were used for generation of activated T and B lymphocytes. M. hyopneumoniae made clusters of spleen cells and also affected the cellular activity and viability of spleen cells by alone or with mitogens. Of particular interest, it induced a significant increase in production of TNF-alpha in ConA- treated spleen cells, meaning T helper 1 response. In addition, cell size and mitochondrial membrane potential of M. hyopneumoniae–treated spleen cells were measured by flow cytometric analysis. M. hyopneumoniae did not affect the cell size by alone, whereas ConA or LPS profoundly increased the cell size. Taken together, M. hyopneumoniae significantly affect the cellular activity and cytokine production of spleen cells by alone or in a combination of ConA. This study provides valuable information for production of the vaccine against M. hyopneumoniae.