Fenbendazole Exhibits Differential Anticancer Effects In Vitro and In Vivo in Models of Mouse Lymphoma.

Fenbendazole (FBZ) has been safely used as an antiparasitic agent in animals for decades, and the anticancer effects of FBZ have been studied through various mechanisms. However, there is a lack of in vivo studies that include lymphoma. Therefore, this study examined the effects of FBZ on EL-4 cells and a mouse T lymphoma model. FBZ induced G2/M phase arrest in EL-4 cells, resulting in cell death and decreased metabolic activity. However, FBZ had no anticancer effects on an EL-4 mouse lymphoma model in vivo, as evident by rapid weight loss and tumor growth comparable to the control. The FBZ-treated EL-4 cells expressed higher levels of PD-L1 and CD86, which are associated with T cell immunity in the tumor microenvironment (TME), than the controls. Furthermore, the hematoxylin and eosin staining of the FBZ-treated tumor tissues showed a starry sky pattern, which is seen in actively proliferating cancer tissues, and an immunohistochemical analysis revealed a high percentage of immunosuppressive M2 macrophages. These changes in the immune activity in the TME contradict the results of the in vitro experiments, and further studies are needed to determine the detailed mechanisms by which FBZ induces these responses.

Dendritic cells resist to disulfiram-induced cytotoxicity, but reduced interleukin-12/23(p40) production.

Disulfiram (DSF), a medication for alcoholism, has recently been used as a repurposing drug owing to its anticancer effects. Despite the crucial role of dendritic cells (DCs) in immune homeostasis and cancer therapy, the effects of DSF on the survival and function of DCs have not yet been studied. Therefore, we treated bone marrow-derived DCs with DSF and lipopolysaccharide (LPS) and performed various analyses. DCs are resistant to DSF and less cytotoxic than bone marrow cells and spleen cells. The viability and metabolic activity of DCs hardly decreased after treatment with DSF in the absence or presence of LPS. DSF did not alter the expression of surface markers (MHC II, CD86, CD40, and CD54), antigen uptake capability, or the antigen-presenting ability of LPS-treated DCs. DSF decreased the production of interleukin (IL)-12/23 (p40), but not IL-6 or tumor necrosis factor-α, in LPS-treated DCs. We considered the granulocyte-macrophage colony-stimulating factor (GM-CSF) as a factor to make DCs resistant to DSF-induced cytotoxicity. The resistance of DCs to DSF decreased when GM-CSF was not given or its signaling was inhibited. Also, GM-CSF upregulated the expression of a transcription factor XBP-1 which is essential for DCs' survival. This study demonstrated for the first time that DSF did not alter the function of DCs, had low cytotoxicity, and induced differential cytokine production.

Immunostimulatory activity and safety evaluation of Bordetella bronchiseptica-derived lipopolysaccharide, a new vaccine adjuvant candidate.

Adjuvants are used to elicit strong immune responses for vaccines that show poor immunogenicity. Previously, we demonstrated that a sonicated bacterin of Bordetella bronchiseptica can be used as a safe adjuvant that enhances the antigen-presenting capability of dendritic cells (DCs). In this study, we purified the lipopolysaccharide (LPS) of B. bronchiseptica (Bb-LPS) and investigated its immunogenic effects on DCs compared to those of Escherichia coli O26:B6 (O26)-derived LPS (O26-LPS), a positive control. Bb-LPS was purified using an LPS extraction kit. Limulus amebocyte lysate assay was performed to determine the optimal concentration of Bb-LPS and O26-LPS for treatment. Bb-LPS increased the metabolic activity of DCs at a concentration of 0 to 250 EU/mL, similar to that of O26-LPS. Bb-LPS significantly increased the expression level of CD40 and CD54, related to the immune responses of DCs. Bb-LPS enhanced the antigen-presenting capability of DCs and significantly increased the interferon-gamma/interleukin-4 ratio of CD4+ T cells co-cultured with DCs to 0.95 (p < 0.05). Moreover, Bb-LPS increased the production of pro-inflammatory cytokines in a safer manner than that obtained by O26-LPS. In vivo safety tests revealed that Bb-LPS was less toxic than O26-LPS in mice. This study demonstrated that Bb-LPS showed unique immune characteristics and immunogenic effects on the antigen-presenting capability of DCs, which differed from those of O26-LPS. This study provides valuable information for basic and clinical research for developing safe vaccine adjuvants.

Sonicated Bordetella bronchiseptica Bacterin Can Protect Dendritic Cells from Differential Cytotoxicity Caused by Doxorubicin and Vincristine and Enhance Their Antigen-Presenting Capability.

Doxorubicin (DOX) and vincristine (VC) are anti-cancer drugs commonly used for lymphoma in veterinary and human medicine. However, there are several side effects caused by these drugs. In this study, the protective effects of sonicated Bordetella bronchiseptica bacterin (sBb) on dendritic cells (DCs) damaged by two anti-cancer drugs were investigated. DCs play important roles in the innate and adaptive immunity of hosts, especially activating T cells that can suppress tumor growth. The metabolic activity of DCs significantly increased after the treatment with sBb compared to that of control DCs. In addition, there was a marked change in mitochondrial integrity between DOX-treated DC and DOX + sBb-treated DCs. Flow cytometric analysis also demonstrated that sBb upregulated the expression of the surface markers of DCs, particularly CD54. In mixed lymphocyte responses, sBb significantly increased the antigen-presenting capability of DCs. In particular, sBb increased the capability of control DCs by approximately 150% and that of VC-treated DCs by 221%. These results suggest that sBb can be used as a potential immunostimulatory agent to protect DCs from anti-cancer drug-induced damage and provide fundamental information about using a combination of DCs and vincristine in immunotherapy.

Evaluation of the effects of disulfiram, an alcohol-aversive agent with anti-cancer activity, on mouse bone marrow cells.

Disulfiram (DSF) is an aldehyde dehydrogenase inhibitor. DSF has potent anti-cancer activity for solid and hematological malignancies. Although the effects on cancer cells have been proven, there have been few studies on DSF toxicity in bone marrow cells (BMs). DSF reduces the metabolic activity and the mitochondrial membrane potential of BMs. In subset analyses, we confirmed that DSF does not affect the proportion of BMs. In addition, DSF significantly impaired the metabolic activity and differentiation of BMs treated with granulocyte macrophage-colony stimulating factor, an essential growth and differentiation factor for BMs. To measure DSF toxicity in BMs in vivo, mice were injected with 50 mg/kg, a dose used for anti-cancer effects. DSF did not significantly induce BM toxicity in mice and may be tolerated by antioxidant defense mechanisms. This is the first study on the effects of DSF on BMs in vitro and in vivo. DSF has been widely studied as an anti-cancer drug candidate, and many anti-cancer drugs lead to myelosuppression. In this regard, this study can provide useful information to basic science and clinical researchers.

ER71/ETV2 Promotes Hair Regeneration from Chemotherapeutic Drug-Induced Hair Loss by Enhancing Angiogenesis.

Chemotherapy-induced alopecia and hair loss can be stressful in patients with cancer. The hair grows back, but sometimes the hair tends to stay thin. Therefore, understanding mechanisms regulating hair regeneration may improve the management of chemotherapy-induced alopecia. Previous studies have revealed that chemotherapeutic agents induce a hair follicle vascular injury. As hair growth is associated with micro-vessel regeneration, we postulated that the stimulation of angiogenesis might enhance hair regeneration. In particular, mice treated with 5-fluorouracil (5-FU) showed delayed anagen initiation and reduced capillary density when compared with untreated controls, suggesting that the retardation of anagen initiation by 5-FU treatment may be attributed to the loss of perifollicular micro-vessels. We investigated whether the ETS transcription factor ETV2 (aka ER71), critical for vascular development and regeneration, can promote angiogenesis and hair regrowth in a 5-FU-induced alopecia mouse model. Tie2-Cre; Etv2 conditional knockout (CKO) mice, which lack Etv2 in endothelial cells, presented similar hair regrowth rates as the control mice after depilation. Following 5-FU treatment, Tie2-Cre; Etv2 CKO mice revealed a significant reduction in capillary density, anagen induction, and hair restoration when compared with controls. Mice receiving lentiviral Etv2 injection after 5-FU treatment showed significantly improved anagen induction and hair regrowth. Two-photon laser scanning microscopy revealed that enforced Etv2 expression restored normal vessel morphology after 5-FU mediated vessel injury. Our data suggest that vessel regeneration strategies may improve hair regrowth after chemotherapeutic treatment.

Bordetella bronchiseptica is a potent and safe adjuvant that enhances the antigen-presenting capability of dendritic cells.

We previously demonstrated that Bordetella bronchiseptica (B. bronchiseptica) antigen (Ag) enhances the Mycoplasma hyopneumoniae Ag-specific immune response. The focus of this study was whether acellular bacterin of B. bronchiseptica could be used as an adjuvant to increase antigen-presenting capability of dendritic cells (DCs) by increasing the level of activation. The metabolic activity of DCs was increased by B. bronchiseptica, similar to lipopolysaccharide (LPS). Flow cytometry analysis revealed that B. bronchiseptica increases the expression of major histocompatibility complex class-2, cluster of differentiation (CD)40, CD54, and CD86 which are closely related to DC-mediated immune responses. B. bronchiseptica enhanced the production of cytokines related to adaptive immune responses. Furthermore, the survival rate of B. bronchiseptica-injected groups was 100% at 15 and 20 mg/kg doses, whereas that of LPS-injected groups was only 20%, 0% at 15 and 20 mg/kg doses respectively, and so B. bronchiseptica is likely to be safer than LPS. Taken together, these results indicate that B. bronchiseptica can be used as an adjuvant to enhance the antigen-presenting capability of DCs. B. bronchiseptica is a candidate for producing vaccines, especially in case of DC-mediating efficacy and safety demands. This study provides researchers and clinicians with valuable information regarding the usage of B. bronchiseptica as a safe bacteria-derived immunostimulating agent for developing efficient vaccines.

Dapsone modulates lipopolysaccharide-activated bone marrow cells by inducing cell death and down-regulating tumor necrosis factor-α production.

Dapsone, an antibiotic, has been used to cure leprosy. It has been reported that dapsone has anti-inflammatory activity in hosts; however, the anti-inflammatory mechanism of dapsone has not been fully elucidated. The present study investigated the anti-inflammatory effects of dapsone on bone marrow cells (BMs), especially upon exposure to lipopolysaccharide (LPS). We treated BMs with LPS and dapsone, and the treated cells underwent cellular activity assay, flow cytometry analysis, cytokine production assessment, and reactive oxygen species assay. LPS distinctly activated BMs with several characteristics including high cellular activity, granulocyte changes, and tumor necrosis factor alpha (TNF-α) production increases. Interestingly, dapsone modulated the inflammatory cells, including granulocytes in LPS-treated BMs, by inducing cell death. While the percentage of Gr-1 positive cells was 57% in control cells, LPS increased that to 75%, and LPS plus dapsone decreased it to 64%. Furthermore, dapsone decreased the mitochondrial membrane potential of LPS-treated BMs. At a low concentration (25 µg/mL), dapsone significantly decreased the production of TNF-α in LPS-treated BMs by 54%. This study confirmed that dapsone has anti-inflammatory effects on LPS-mediated inflammation via modulation of the number and function of inflammatory cells, providing new and useful information for clinicians and researchers.

Bordetella bronchiseptica antigen enhances the production of Mycoplasma hyopneumoniae antigen-specific immunoglobulin G in mice.

We previously demonstrated that Bordetella (B.) bronchiseptica antigen (Ag) showed high immunostimulatory effects on mouse bone marrow cells (BMs) while Mycoplasma (M.) hyopneumoniae Ag showed low effects. The focus of this study was to determine if B. bronchiseptica Ag can enhance the M. hyopneumoniae Ag-specific immune response and whether the host's immune system can recognize both Ags. MTT assay results revealed that each or both Ags did not significantly change BM metabolic activity. Flow cytometry analysis using carboxyfluorescein succinimidyl ester showed that B. bronchiseptica Ag can promote the division of BMs. In cytokine and nitric oxide (NO) assays, B. bronchiseptica Ag boosted production of tumor necrosis factor-alpha in M. hyopneumoniae Ag-treated BMs, and combined treatment with both Ags elevated the level of NO in BMs compared to that from treatment of M. hyopneumoniae Ag alone. Immunoglobulin (Ig)G enzyme-linked immunosorbent assay using the sera of Ag-injected mice clearly indicated that B. bronchiseptica Ag can increase the production of M. hyopneumoniae Ag-specific IgG. This study provided information valuable in the development of M. hyopneumoniae vaccines and showed that B. bronchiseptica Ag can be used both as a vaccine adjuvant and as a vaccine Ag.

Paclitaxel inhibits the hyper-activation of spleen cells by lipopolysaccharide and induces cell death.

Paclitaxel was isolated from the bark of the Pacific yew, Taxus brevifolia, and used as an anticancer agent. Paclitaxel prevents cancer cell division by inhibiting spindle fiber function, inducing cell death. A recent study demonstrated that paclitaxel binds to myeloid differentiation protein-2 of Toll-like receptor 4 and prevents the signal transduction of lipopolysaccharide (LPS). Paclitaxel converts immune cells hypo-responsive to LPS. In this study, we investigated whether paclitaxel can inhibit the phenotype and function of immune cells. To accomplish this, we used spleen cells, a major type of immune cell, LPS, a representative inflammatory agent and a mitogen for B lymphocytes. LPS profoundly increased the activation and cytokine production of spleen cells. However, paclitaxel significantly inhibited LPS-induced hyper-activation of spleen cells. Furthermore, we found that paclitaxel induced cell death of LPS-treated spleen cells. These results suggest that paclitaxel can inhibit the hyper-immune response of LPS in spleen cells via a variety of mechanisms. These findings suggest that paclitaxel can be used as a modulating agent for diseases induced by hyper-activation of B lymphocytes. Taken together, these results demonstrate that paclitaxel inhibits the function of spleen cells activated by LPS, and further induces cell death.

Evaluation of adjuvant effects of fucoidan for improving vaccine efficacy.

Fucoidan is a sulfated polysaccharide derived from brown seaweed, including Fucus vesiculosus. This compound is known to have immunostimulatory effects on various types of immune cells including macrophages and dendritic cells. A recent study described the application of fucoidan as a vaccine adjuvant. Vaccination is regarded as the most efficient prophylactic method for preventing harmful or epidemic diseases. To increase vaccine efficacy, effective adjuvants are needed. In the present study, we determined whether fucoidan can function as an adjuvant using vaccine antigens. Flow cytometric analysis revealed that fucoidan increases the expression of the activation markers major histocompatibility complex class II, cluster of differentiation (CD)25, and CD69 in spleen cells. In combination with Bordetella bronchiseptica antigen, fucoidan increased the viability and tumor necrosis factor-α production of spleen cells. Furthermore, fucoidan increased the in vivo production of antigen-specific antibodies in mice inoculated with Mycoplasma hyopneumoniae antigen. Overall, this study has provided valuable information about the use of fucoidan as a vaccine adjuvant.

Differential effects of fucoidans with low and high molecular weight on the viability and function of spleen cells.

Fucoidan is an edible sulfated polysaccharide purified from brown algae that has multiple biological activities. However, the effects of fucoidans of different molecular weights on immune cells have not been determined. Thus, we treated spleen cells with low- and high-molecular-weight fucoidans (LMF and HMF, respectively). Viability assays demonstrated that HMF enhanced the viability and prevented the death of spleen cells. Furthermore, functional analysis revealed that HMF significantly increased the production of interferon-γ and nitric oxide. In contrast, LMF had low activity and was relatively toxic to spleen cells. Taken together, these results indicate that HMF makes the greatest contribution to the immunostimulatory activity of fucoidan mixtures. Additionally, fucoidans with different molecular weights may have different effects on the viability and function of immune cells. This study increases our understanding of fucoidans, and may broaden their use in the basic research and clinical fields.

Possible involvement of galectin-3 in microglial activation in the hippocampus with trimethyltin treatment.

Trimethyltin (TMT) is an organotin neurotoxicant with effects that are selectively localized to the limbic system (especially the hippocampus), which produces memory deficits and temporal lobe seizures. Galectin-3 (Gal-3) is a beta-galactoside-binding lectin that is important in cell proliferation and regulation of apoptosis. The present study evaluated the temporal expression of Gal-3 in the hippocampus of adult BALB/c mice after TMT treatment (i.p., 2.5mg/kg). Western blotting analyses showed that Gal-3 immunoreactivity began to increase days after treatment; the immunoreactivity peaked significantly within days after treatment but significantly declined between days 4 and 8. Immunohistochemical analysis indicated that Gal-3 expression was very rare in the hippocampi of vehicle-treated controls. However, Gal-3 immunoreactivity appeared between 2 and 8 days after TMT treatment and was primarily localized to the hippocampal dentate gyrus (DG), in which neuronal degeneration occurred. The immunoreactivity was detected predominantly in most of the Iba1-positive microglia and in some GFAP-positive astrocytes of the hippocampal DG. Furthermore, Gal-3 expression co-localized with the pro-inflammatory enzymes cyclooxygenase-2 and inducible nitric oxide synthase in the hippocampal DG. Therefore, we suggest that Gal-3 is involved in the inflammatory process of neurodegenerative disorder induced by organotin intoxication.

Immunostimulatory Effects of ß-glucan Purified from Paenibacillus polymyxa JB115 on Mouse Splenocytes.

We investigated the effects of ß-glucan purified from Paenibacillus polymyxa JB115 on the viability and proliferation of splenocytes. Splenocytes play a critical role in host immunity. MTT assays and trypan blue exclusion tests revealed that ß-glucan significantly promoted the viability and proliferation of splenocytes over a range of concentrations. However, there was no specific subset change. ß-glucan protected splenocytes from cytokine withdrawal-induced spontaneous cell death. For further mechanistic studies, ELISA assay revealed that ß-glucan enhanced the expression of anti-apoptotic molecules and interleukin 7 (IL-7), a cytokine critical for lymphocyte survival. We also investigated the IL-2 dependency of ß-glucan-treated splenocytes to determine if treated cells could still undergo clonal expansion. In flow cytometric analysis, ß-glucan induced increased levels of the activation marker CD25 on the surface of splenocytes and ß-glucan-treated splenocytes showed higher proliferation rates in response to IL-2 treatment. This study demonstrates that ß-glucan can enhance the survival of splenocytes and provides valuable information to broaden the use of ß-glucan in research fields.

Cytoprotective effects of fucoidan, an algae-derived polysaccharide on 5-fluorouracil-treated dendritic cells.

Although chemotherapeutic anticancer agents are effective, they also attack normal immune cells due to a lack of selectivity. 5-Fluorouracil (5-FU) is a representative anticancer agent that induces immunosuppression in cancer patients as a side effect. Fucoidan is an algae-derived sulfated polysaccharide that has recently been recognized as a hematopoietic mobilizer and immunomodulator. In this study, we investigated the cytoprotective effect of fucoidan on dendritic cells (DCs) against 5-FU-induced cellular damage. Several kinds of assays including flow cytometric analysis demonstrated the cytoprotective efficacy of fucoidan. In addition, fucoidan increased the expression of immune-related surface markers on and the alloproliferative capacity of DCs exposed to 5-FU. For investigating action mechanism, the expression levels of apoptosis-related molecules were measured. Taken together, the results of this study suggest that fucoidan, a marine-derived polysaccharide, has cytoprotective effects on DCs, the most potent antigen-presenting cell type, against 5-FU-induced cellular damage. These results provide valuable information to use fucoidan as an immunostimulatory agent for the chemotherapy of cancer patients.

Galectin-3 immunohistochemistry in the vomeronasal organ of the domestic pig, Sus scrofa.

The immunohistochemical localization of galectin-3, a ß-galactoside-binding protein, was studied in the vomeronasal organ (VNO) of fetal, 1-day-old, and 6-month-old pigs. In all age groups, the porcine VNO consisted of vomeronasal sensory epithelium (VSE) located medially and non-sensory vomeronasal respiratory epithelium (VRE) located laterally. In the pig, the VNO epithelium increased in height with postnatal development from fetus to adult. In the VSE of all stages examined, galectin-3 immunostaining was seen in the supporting cells and free border, but not in receptor or basal cells. Galectin-3 immunostaining was seen in all layers of the VRE, and the intensity increased with postnatal development. In the lamina propria, galectin-3 was detected in some ductal epithelial cells and the vomeronasal nerve sheath, but not in the acini of the Jacobson glands in all age groups. In view of these observations, we postulate that galectin-3 plays a role in cell survival and cell adhesion in both the VSE and VRE of porcine VNO in all age groups.

Fucoidan enhances the survival and sustains the number of splenic dendritic cells in mouse endotoxemia.

Fucoidan is a sulfated polysaccharide derived from brown algae that has been reported to perform multiple biological activities, including immunostimulation. In this study, we investigated whether fucoidan has beneficial effects on endotoxemia induced by LPS, a septic model in mice. The focus of this study was on survival rates and spleen function of the mice upon treatment. We found that fucoidan had prophylactic effects on the survival rate of mice with endotoxemia. Flow cytometric analysis using antibodies for subset-specific markers revealed that fucoidan profoundly reversed the depleted population of dendritic cells in mice with endotoxemia. According to Western blot analysis, the spleen cells of LPS/fucoidan-treated mice showed a higher expression of anti-apoptotic molecules compared to those of LPS-treated mice. Also, fucoidan-treated spleen cells were more responsive to mitogens. Taken together, these results demonstrate that fucoidan pre-treatment has beneficial effects on the survival rate and function of the spleen in mice with endotoxemia. This study may broaden the use of fucoidan in clinical fields, especially endotoxemia.

Maturation of bone marrow-derived dendritic cells by a novel ß-glucan purified from Paenibacillus polymyxa JB115.

We investigated the immunostimulatory effects of a novel ß-glucan purified from Paenibacillus (P.) polymyxa JB115 on bone marrow-derived dendritic cells (DCs), a type of potent antigen-presenting cells. ß-glucan isolated from P. polymyxa JB115 enhanced the viability and induced the maturation of DCs. ß-glucan markedly increased the cytokine production of DCs and surface expression of DC markers. In addition, DCs treated with ß-glucan showed a higher capacity to stimulate allogeneic spleen cell proliferation compared to those treated with medium alone. These results demonstrate the effect of ß-glucan on DC maturation and may increase the use of ß-glucan.

Diphlorethohydroxycarmalol, isolated from the brown algae Ishige okamurae, protects against radiation-induced cell damage in mice.

The aim of this study was to evaluate the radioprotective effects of diphlorethohydroxycarmalol (DPHC), isolated from the brown algae Ishige okamurae, in mice subjected to gamma irradiation. DPHC significantly decreased the level of radiation-induced intracellular reactive oxygen species in cultured Chinese hamster lung fibroblast (V79-4) cells (p < 0.05), enhanced cell viability that decreased after exposure to γ-rays, and reduced radiation-induced apoptosis in the V79-4 cells. Pretreatment with DPHC (100 mg/kg) in mice prior to irradiation significantly protected the intestinal crypt cells in the jejunum (p < 0.01) and maintained villi height (p < 0.01), compared with those of the vehicle-treated irradiated group. Mice pretreated with DPHC also exhibited dose-dependent increases in the bone marrow cell viability. The dose-reduction factor for gamma irradiation in the DPHC-pretreated mice was 2.05 at 3.5 days after irradiation. These results suggest that DHPC plays a role in protecting cells from irradiation-induced apoptosis, through the scavenging of reactive oxygen species in vitro, and that DPHC significantly protected intestinal progenitor cells and bone marrows cells that were decreased by gamma irradiation in vivo.

Stimulatory effects of ginsan on the proliferation and viability of mouse spleen cells.

Ginsan is an acidic polysaccharide purified from Panax ginseng, a famous oriental herb. Although a variety of biological activities of ginsan have been studied, the effects of ginsan on spleen cells are not fully elucidated. We investigated the effect of ginsan on the viability and proliferation of spleen cells. Using Cell Counting Kit-8(R) solution and trypan blue solution, we found that ginsan significantly enhanced viability and proliferation. Multiple clusters, indicating proliferation, were observed in ginsan-treated spleen cells and, carboxyfluorescein succinimidyl ester and surface marker staining assay revealed that ginsan promoted proliferation from CD19(+) B cells rather than CD4(+) or CD8(+) T cells. In addition, ginsan decreased the percentage of late apoptotic cells. Ginsan increased the surface expression of CD25 and CD69 as well as production of interleukin-2 from spleen cells, suggesting increased activation. Taken together, these results demonstrate that ginsan increases the viability and proliferation of spleen cells via multiple mechanisms, valuable information for broadening the use of ginsan in clinical and research settings.