Screening for Active Compounds Targeting Human Natural Killer Cell Activation Identifying Daphnetin as an Enhancer for IFN-γ Production and Direct Cytotoxicity.

Natural killer (NK) cells are a potent weapon against tumor and viral infection. Finding active compounds with the capacity of enhancing NK cell effector functions will be effective to develop new anti-cancer drugs. In this study, we initially screened 287 commercially available active compounds by co-culturing with peripheral blood mononuclear cells (PBMCs). We found that five compounds, namely, Daphnetin, MK-8617, LW6, JIB-04, and IOX1, increased the IFN-γ+ NK cell ratio in the presence of IL-12. Further studies using purified human primary NK cells revealed that Daphnetin directly promoted NK cell IFN-γ production in the presence of IL-12 but not IL-15, while the other four compounds acted on NK cells indirectly. Daphnetin also improved the direct cytotoxicity of NK cells against tumor cells in the presence of IL-12. Through RNA-sequencing, we found that PI3K-Akt-mTOR signaling acted as a central pathway in Daphnetin-mediated NK cell activation in the presence of IL-12. This was further confirmed by the finding that both inhibitors of PI3K-Akt and its main downstream signaling mTOR, LY294002, and rapamycin, respectively, can reverse the increase of IFN-γ production and cytotoxicity in NK cells promoted by Daphnetin. Collectively, we identify a natural product, Daphnetin, with the capacity of promoting human NK cell activation via PI3K-Akt-mTOR signaling in the presence of IL-12. Our current study opens up a new potential application for Daphnetin as a complementary immunomodulator for cancer treatments.

Dietary source of polyunsaturated fatty acids influences cell cytotoxicity in broiler chickens.

The current study aims to investigate the effects of dietary source of n-3 polyunsaturated fatty acids (PUFA) on immune response in broiler chickens, represented by cytotoxic cell activity. A total of 255 one-day-old male Cobb 500 broiler chickens were fed on fish oil (FO)-, flaxseed oil-enriched diets at 50 and 19 g/kg, respectively, in addition to the soybean-based control diet. At slaughter, samples of blood and spleen were harvested from 20 birds/treatment (n = 20). The immune tissues' fatty acid profile was analyzed by gas chromatography, and the cytotoxic cell activity was investigated. The results showed that supplementing broiler chickens with diets rich in n-3 PUFA had a substantial effect on the broiler immune tissues' fatty acid profile. Cytotoxic cell activity was significantly higher in splenocytes and peripheral blood mononuclear cells (PBMCs) from broilers fed flaxseed oil than those provided FO and the soybean control diet. These results suggest that flaxseed oil may be used to enrich chickens with n-3 PUFA and improve the immune status of chicken flocks to resist diseases.

Aerosol 1,25-dihydroxyvitamin D3 supplementation: A strategy to boost anti-tumor innate immune activity.

BACKGROUND: 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] plays a role in calcium homeostasis but can also exert immunomodulatory effects. In lungs, characterized by a particular immunosuppressive environment primarily due to the presence of alveolar macrophages (AM), 1,25(OH)2D3 has been shown to favor the immune response against pathogens. Here, we explored the ability of aerosolized 1,25(OH)2D3 to locally promote an anti-tumor phenotype in alveolar macrophages (AM) in the treatment of lung metastases. METHODS: Cytotoxicity assay has been used to assess the capability of AM, in vitro treated of not with 1,25(OH)2D3, to stimulate NK cells. Sulforhodamine B (SRB) assay has been used to assess the effect of 1,25(OH)2D3 on MC-38 and B16 tumor cells in vitro growth. 1,25(OH)2D3 was aerosolized in immunocompetent mouse models to evaluate the effect of local administration of 1,25(OH)2D3 on in vivo growth of MC-38 and B16 tumor cells within lungs and on infiltrating immune cells. RESULTS: In vitro incubation of naïve AM with 1,25(OH)2D3 improved their ability to stimulate NK cell cytotoxicity. In vivo aerosolized 1,25(OH)2D3 significantly reduced the metastatic growth of MC-38 colon carcinoma, a tumor histotype that frequently metastasizes to lung in human. Immune infiltrate obtained from digested lungs of 1,25(OH)2D3-treated mice bearing MC-38 metastases revealed an increased expression of MHCII and CD80 on AM and an up-modulation of CD69 expression on effector cells that paralleled a strong increased ability of these cells to kill MC-38 tumor in vitro. CONCLUSIONS: Together, these data show that aerosol delivery can represent a feasible and novel approach to supplement 1,25(OH)2D3 directly to the lungs promoting the activation of local immunity against cancer.

Immunogenicity and Toxicity of Different Adjuvants Can Be Characterized by Profiling Lung Biomarker Genes After Nasal Immunization.

The efficacy of vaccine adjuvants depends on their ability to appropriately enhance the immunogenicity of vaccine antigens, which is often insufficient in non-adjuvanted vaccines. Genomic analyses of immune responses elicited by vaccine adjuvants provide information that is critical for the rational design of adjuvant vaccination strategies. In this study, biomarker genes from the genomic analyses of lungs after priming were used to predict the efficacy and toxicity of vaccine adjuvants. Based on the results, it was verified whether the efficacy and toxicity of the tested adjuvants could be predicted based on the biomarker gene profiles after priming. Various commercially available adjuvants were assessed by combining them with the split influenza vaccine and were subsequently administered in mice through nasal inoculation. The expression levels of lung biomarker genes within 24 h after priming were analyzed. Furthermore, we analyzed the antibody titer, cytotoxic T lymphocyte (CTL) induction, IgG1/IgG2a ratio, leukopenic toxicity, and cytotoxicity in mice vaccinated at similar doses. The association between the phenotypes and the changes in the expression levels of biomarker genes were analyzed. The ability of the adjuvants to induce the production of antigen-specific IgA could be assessed based on the levels of Timp1 expression. Furthermore, the expression of this gene partially correlated with the levels of other damage-associated molecular patterns in bronchoalveolar lavage fluid. Additionally, the changes in the expression of proteasome- and transporter-related genes involved in major histocompatibility complex class 1 antigen presentation could be monitored to effectively assess the expansion of CTL by adjuvants. The monitoring of certain genes is necessary for the assessment of leukopenic toxicity and cytotoxicity of the tested adjuvant. These results indicate that the efficacy and toxicity of various adjuvants can be characterized by profiling lung biomarker genes after the first instance of immunization. This approach could make a significant contribution to the development of optimal selection and exploratory screening strategies for novel adjuvants.

Andiroba oil (Carapa guianensis Aubl) shows cytotoxicity but no mutagenicity in the ACPP02 gastric cancer cell line.

Andiroba (Carapa guianensis Aubl) is an Amazonian plant whose oil has been widely used in traditional medicine for various purposes, including anti-inflammation. Research reports indicate that the oil can confer antitumor activity due to the presence of fatty acids, which can directly influence cell death mechanisms. Thus, andiroba oil (AO) has gained interest for its potential to be used in antineoplastic therapies. Here, we report an in vitro analysis of the cytotoxic and mutagenic potential of AO in the gastric cancer cell line, ACP02. Cell survival was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, differential staining with ethidium bromide and acridine orange assessed apoptosis-necrosis, and mutagenesis was assessed by the micronucleus test. The apolar oil was first diluted in 0.1% dimethyl sulfoxide (DMSO) and then further diluted to six concentrations (0.01, 0.1, 1, 10 and 100 µg/mL and 1 mg/mL) in RPMI medium. Controls included RPMI alone (negative control) and 0.1% DMSO diluted in medium (vehicle control). The MTT test showed that AO significantly reduced cell viability (P < .05) only when the highest tested concentration was applied for 48 hours. The apoptosis/necrosis test showed that the highest concentration of AO induced cell death by apoptosis at 24 and 48 hours. There was no statistically significant increase in the frequency of micronuclei. The ability of the AO to decrease the viability of ACP02 cells via apoptosis, without exerting mutagenic effects, suggests that the oil could be useful as an alternative therapeutic agent for primary tumors of stomach cancer.

Developing a 3D B Cell Lymphoma Culture System to Model Antibody Therapy.

Diffuse large cell B cell lymphoma (DLBCL) accounts for approximately 30%-40% of all non-Hodgkin lymphoma (NHL) cases. Current first line DLBCL treatment results in long-term remission in more than 60% of cases. However, those patients with primary refractory disease or early relapse exhibit poor prognosis, highlighting a requirement for alternative therapies. Our aim was to develop a novel model of DLBCL that facilitates in vitro testing of current and novel therapies by replicating key components of the tumor microenvironment (TME) in a three-dimensional (3D) culture system that would enable primary DLBCL cell survival and study ex vivo. The TME is a complex ecosystem, comprising malignant and non-malignant cells, including cancer-associated fibroblasts (CAF) and tumor-associated macrophages (TAM) whose reciprocal crosstalk drives tumor initiation and growth while fostering an immunosuppressive milieu enabling its persistence. The requirement to recapitulate, at least to some degree, this complex, interactive network is exemplified by the rapid cell death of primary DLBCL cells removed from their TME and cultured alone in vitro. Building on previously described methodologies to generate lymphoid-like fibroblasts from adipocyte derived stem cells (ADSC), we confirmed lymphocytes, specifically B cells, interacted with this ADSC-derived stroma, in the presence or absence of monocyte-derived macrophages (MDM), in both two-dimensional (2D) cultures and a 3D collagen-based spheroid system. Furthermore, we demonstrated that DLBCL cells cultured in this system interact with its constituent components, resulting in their improved viability as compared to ex-vivo 2D monocultures. We then assessed the utility of this system as a platform to study therapeutics in the context of antibody-directed phagocytosis, using rituximab as a model immunotherapeutic antibody. Overall, we describe a novel 3D spheroid co-culture system comprising key components of the DLBCL TME with the potential to serve as a testbed for novel therapeutics, targeting key cellular constituents of the TME, such as CAF and/or TAM.

Polysaccharides from Hedyotis diffusa enhance the antitumor activities of cytokine-induced killer cells.

Hedyotis diffusa is a well-known traditional Chinese herbal medicine. The polysaccharides extracted from H. diffusa (HDP) exhibit a range of pharmacological activities. Transfusion of cytokine-induced killer (CIK) cell is one type of adoptive cellular immunotherapy, which is becoming an important method of cancer immunotherapy. In this present study, we investigate the immunostimulatory effect of HDP on CIK cells. CIK cells were generated by culturing and stimulating peripheral blood monocytes of healthy volunteers. They were treated with HDP at three different concentrations (10, 50, and 100 µg/mL). The effect of HDP on CIK cell populations, intracellular cytokine production, and apoptosis was examined by flow cytometry. The antitumor effect of HDP on CIK cells was determined by cytotoxicity assay. Furthermore, the effect of HDP on the antitumor activity of CIK cells in a mouse model was investigated. HDP increased the percentage of CD3+CD56+ CIK cells but did not significantly change the percentage of CD4+, CD8+, or CD4+CD25+ CIK cells. The HDP-treated CIK cells showed a greater ability to kill tumor cells, as well as higher production of interferon-γ and tumor necrosis factor-α, compared with the no-HDP-treated CIK cells. The HDP-treated CIK cells also found a lower apoptosis level in vitro. Moreover, HDP combined with CIK cells had a stronger inhibitory effect on tumor growth in the mouse model compared with the CIK or HDP treatment alone. In conclusion, the results indicated that HDP enhanced the antitumor activity of CIK cells and could be used for cancer immunotherapy combined with CIK cell therapy.

Selenium-containing ruthenium complex synergizes with natural killer cells to enhance immunotherapy against prostate cancer via activating TRAIL/FasL signaling.

Natural killer (NK) cells-based therapy has been used widely for cancer treatment in clinic trails. However, the immunotherapeutic efficacy of this method has been greatly hindered by tumor evasion and diminished activities of NK cells. In the present study, a selenium (Se)-bearing ruthenium (Ru) complex (RuSe) was designed that could synergistically potentiate NK cell-mediated killing against prostate cancer cells. As expected, pretreatment of cancer cells with subtoxic doses of RuSe effectively augmented the lysis potency of NK cells, with up to 2.46-fold enhancement than NK cells alone, against PC3 cells. More importantly, low concentrations of RuSe could augment the tumor destroying potency of NK cells derived from 10 clinical patients, with the enhancement range from 0.78- to 11.9-fold against PC3 cells and 0.67- to 3.8-fold against LNCAP cells. Mechanistic studies revealed that the sensitizing effect of RuSe primarily depended on TRAIL/TRAIL-R and Fas/FasL-mediated signaling. Furthermore, the increased expression level of these ligands highly relied on ROS overproduction-triggered DNA damage and the downstream ATM and ATR pathways. Furthermore, RuSe potently activated and synergized with NK cells to restrain tumor growth in vivo without causing toxic side effects on major organs. Taken together, the current study not only provides a strategy for application of metal complexes in chemo-immunotherapy but also sheds light on the potential roles and mechanisms of action on such Se-containing drugs as efficient immune-sensitizing agents for NK cell-based immunotherapy.

Immunomodulatory and Metabolic Changes after Gnetin-C Supplementation in Humans.

Gnetin-C is a naturally occurring stilbene derived from the seeds of Gnetum gnemon L., an edible plant native to Southeast Asia that is called melinjo. Although the biological properties and safety of G. gnemon extract, which contains nearly 3% Gnetin-C, have been confirmed in various human studies, whether or not pure Gnetin-C is safe for humans is unclear at present. We conducted a randomized, double-blind, placebo-controlled trial. Healthy subjects were randomly divided into two groups. The interventional group (n = 6) was given Gnetin-C, and the control group (n = 6) was provided a placebo, for 14 days. Lipid profiles, biomarkers of oxidative stress and circulating blood cells were assessed before and after the intervention. All subjects completed the study, with no side effects reported across the study duration. Gnetin-C supplementation demonstrated a statistically significant increase in the absolute number of circulating natural killer (NK) cells expressing the activating receptors NKG2D and NKp46. NK cells derived from subjects who received Gnetin-C for two weeks showed higher cytotoxicity against K562 target cells than those before receiving Gnetin-C. In addition, Gnetin-C also resulted in a significant decrease in the absolute neutrophil count in the blood compared with the placebo. Furthermore, Gnetin-C significantly reduced the levels of uric acid, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, total adiponectin, and high-molecular-weight adiponectin. These data indicate that Gnetin-C has biological effects of enhancing the NK activity on circulating human immune cells. The immunomodulatory effects are consistent with a putative improvement in cancer immunosurveillance via the upregulation of the NKG2D receptor. The study was registered with UMIN-CTR, number 000030364, on 12 December 2017.

Korean mistletoe lectin enhances natural killer cell cytotoxicity via upregulation of perforin expression.

BACKGROUND: Natural killer (NK) cells are crucial components of the innate immune system, providing the first line of defense against pathogens. In a previous study, we demonstrated prophylactic activity of water extract of Korean mistletoe (Viscum album coloratum) on tumor metastasis. However, the leading compound from water extract of Korean mistletoe was not clearly addressed. OBJECTIVE: The purpose of this research was mainly focused on addressing the effect of Korean mistletoe lectin (KMLC) on NK cell cytotoxicity, and the ability of cytokine secretion as well as its signal transduction, mitogen-activated protein kinase (MAPK) pathway. METHODS: KMLC was used to test NK cell-mediated cytotoxicity in vitro and in vivo. Non-isotope cytotoxicity assay (bis-N,N,N',N'-tetraacetic acid (BATDA) release assay) was performed to test the cytotoxicity of NK cells against target tumor cells. Receptor expression was checked by flow cytometry analysis and MAPK signal molecules were analyzed by immunoblotting. RESULTS AND CONCLUSIONS: KMLC at 200 ng/mL increased the cytotoxicity of NK92 cells by 35% compared with untreated cells. KMLC-treated (at 100 ng/mL) mice splenocytes showed a 20% increase in cytotoxic activity. Also, the B chain, one of the subchains of KMLC, increases perforin expression. We demonstrated that the signal transduction controlling NK cell cytotoxicity was mediated by upregulation of the NKG2D receptor and expression of a cytotoxic effector molecule. These results suggested that KMLC possessed immunological activity, mediated by NK cell activation.

A water-soluble derivative of propolis augments the cytotoxic activity of natural killer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Propolis, a resinous material collected from numerous plants by honeybees, has historically been used as a health-promoting food. Recently, due to its potential anti-tumor effects, use of propolis has been proposed as an adjuvant therapy to chemotherapy; however, the effects of propolis on immune responses remain unclear. AIM OF THE STUDY: In this study, we examined the effects of the oral ingestion of propolis on natural killer (NK) cell activity, which is important in immune surveillance against cancer and viral infections. In addition, we assessed the effects of the major components of the water-soluble powder derivative of propolis (WPP). MATERIALS AND METHODS: C57BL/6 (B6) wild-type (WT) and RAG 2-deficient (RAG-/-) mice and BALB/c WT, interferon (IFN)-γ-deficient (IFN-γ-/-), IFN-γ receptor-deficient (IFN-γR-/-) and RAG-/- mice were orally administered WPP or its major components. NK cell populations and cytotoxic activity were then examined by flow cytometry and 51Cr release assay, respectively. RESULTS: While the cytotoxic activity of NK cells was increased following administration of 100 mg/kg/day of WPP for 7 days or 200 or 500 mg/kg/day of WPP for 4 days in WT mice, the proportions of NK cell populations were unaltered. Similar activation of NK cell cytotoxicity was observed when RAG-/-, but not IFN-γ-/- or IFN-γR-/-, mice were orally administered 200 mg/kg/day of WPP for 4 days. Oral ingestion of artepillin C or p-coumaric acid, but not drupanin, augmented NK cell cytotoxicity in a manner similar to WPP and to the mixture of these three components. CONCLUSION: These results suggest that oral ingestion of WPP enhances NK cell cytotoxic activity, but not proliferation, in a manner dependent on IFN-γ and without the contribution of acquired immune responses. Further, artepillin C or p-coumaric acid, but not drupanin, may be the components responsible for this augmentation of NK cell cytotoxicity. These findings suggest the possible utility of WPP as a therapeutic for prevention of cancer development and against viral infection through NK cell activation.

Oral supplementation with liposomal glutathione elevates body stores of glutathione and markers of immune function.

BACKGROUND/OBJECTIVES: Glutathione (GSH) is the most abundant endogenous antioxidant and a critical regulator of oxidative stress. Maintenance of optimal tissues for GSH levels may be an important strategy for the prevention of oxidative stress-related diseases. We investigated if oral administration of liposomal GSH is effective at enhancing GSH levels in vivo. SUBJECTS/METHODS: A 1-month pilot clinical study of oral liposomal GSH administration at two doses (500 and 1000 mg of GSH per day) was conducted in healthy adults. GSH levels in whole blood, erythrocytes, plasma and peripheral blood mononuclear cells (PBMCs) were assessed in 12 subjects at the baseline and after 1, 2 and 4 weeks of GSH administration. RESULTS: GSH levels were elevated after 1 week with maximum increases of 40% in whole blood, 25% in erythrocytes, 28% in plasma and 100% in PBMCs occurring after 2 weeks (P<0.05). GSH increases were accompanied by reductions in oxidative stress biomarkers, including decreases of 35% in plasma 8-isoprostane and 20% in oxidized:reduced GSH ratios (P<0.05). Enhancements in immune function markers were observed with liposomal GSH administration including Natural killer (NK) cell cytotoxicity, which was elevated by up to 400% by 2 weeks (P<0.05), and lymphocyte proliferation, which was elevated by up to 60% after 2 weeks (P<0.05). Overall, there were no differences observed between dose groups, but statistical power was limited due to the small sample size in this study. CONCLUSIONS: Collectively, these preliminary findings support the effectiveness of daily liposomal GSH administration at elevating stores of GSH and impacting the immune function and levels of oxidative stress.

T cell inhibition by pogostone from Pogostemon cablin (Blanco) Benth: In vitro and in vivo immunosuppressive analysis.

Various plant-derived compounds exhibit immunosuppressive activity in pre­clinical investigations, suggesting that they may serve as natural alternatives for the prevention of inflammatory disorders and autoimmune diseases. The aim of the current study was to explore the immunosuppressive potential of pogostone (PO) derived from Pogostemon cablin (Blanco) Benth. Carboxyfluorescein diacetate succinimidyl ester­labeled cell tracking demonstrated that PO (20­80 µM) inhibited Concanavalin A (ConA)­stimulated lymphocyte proliferation, which was mediated by G0/G1 phase arrest and accompanied by significant decreases in the expression of CD69 (early­stage activation marker) and CD25 (mid­stage activation marker) in T cells, as indicated by flow cytometry analysis. Furthermore, the proliferation blocking ability of PO (5­80 µM) was not associated with cytotoxicity in normal lymphocytes or apoptosis in ConA­stimulated lymphocytes. The inflammatory cytokine profile determination using a cytometric beads assay revealed that PO inhibited release of anti­inflammatory interleukin (IL)­10 and pro­inflammatory IL­6 from the stimulated lymphocytes. Furthermore, PO (10, 20 or 40 mg/kg) ameliorated the T­cell mediated delayed type hypersensitivity response in Balb/c mice by reducing leukocyte infiltration and tissue edema, providing a further validation of the direct immunosuppressive activity of PO. Together, the present data suggest that PO would suppress T cell response via a direct non­cytotoxic inactivation at the early stage, accompanied by regulation of the inflammatory cytokine profile, which highlights clinical implications for treatment of immune-based disorders.

Potentiated virucidal activity of pomegranate rind extract (PRE) and punicalagin against Herpes simplex virus (HSV) when co-administered with zinc (II) ions, and antiviral activity of PRE against HSV and aciclovir-resistant HSV.

BACKGROUND: There is a clinical need for new therapeutic products against Herpes simplex virus (HSV). The pomegranate, fruit of the tree Punica granatum L, has since ancient times been linked to activity against infection. This work probed the activity of pomegranate rind extract (PRE) and co-administered zinc (II) ions. MATERIALS AND METHODS: PRE was used in conjunction with zinc (II) salts to challenge HSV-1 and aciclovir-resistant HSV in terms of virucidal plaque assay reduction and antiviral activities in epithelial Vero host cells. Cytotoxicity was determined by the MTS assay using a commercial kit. RESULTS: Zinc sulphate, zinc citrate, zinc stearate and zinc gluconate demonstrated similar potentiated virucidal activity with PRE against HSV-1 by up to 4-fold. A generally parabolic relationship was observed when HSV-1 was challenged with PRE and varying concentrations of ZnSO4, with a maximum potentiation factor of 5.5. Punicalagin had 8-fold greater virucidal activity than an equivalent mass of PRE. However, antiviral data showed that punicalagin had significantly lower antiviral activity compared to the activity of PRE (EC50 = 0.56 µg mL-1) a value comparable to aciclovir (EC50 = 0.18 µg mL-1); however, PRE also demonstrated potency against aciclovir-resistant HSV (EC50 = 0.02 µg mL-1), whereas aciclovir showed no activity. Antiviral action of PRE was not influenced by ZnSO4. No cytotoxicity was detected with any test solution. CONCLUSIONS: The potentiated virucidal activity of PRE by coadministered zinc (II) has potential as a multi-action novel topical therapeutic agent against HSV infections, such as coldsores.

Bitter Melon Enhances Natural Killer-Mediated Toxicity against Head and Neck Cancer Cells.

Natural killer (NK) cells are one of the major components of innate immunity, with the ability to mediate antitumor activity. Understanding the role of NK-cell-mediated tumor killing in controlling of solid tumor growth is still in the developmental stage. We have shown recently that bitter melon extract (BME) modulates the regulatory T cell (Treg) population in head and neck squamous cell carcinoma (HNSCC). However, the role of BME in NK-cell modulation against HNSCC remains unknown. In this study, we investigated whether BME can enhance the NK-cell killing activity against HNSCC cells. Our results indicated that treatment of human NK-cell line (NK3.3) with BME enhances ability to kill HNSCC cells. BME increases granzyme B accumulation and translocation/accumulation of CD107a/LAMP1 in NK3.3 cells exposed to BME. Furthermore, an increase in cell surface expression of CD16 and NKp30 in BME-treated NK3.3 cells was observed when cocultured with HNSCC cells. Collectively, our results demonstrated for the first time that BME augments NK-cell-mediated HNSCC killing activity, implicating an immunomodulatory role of BME. Cancer Prev Res; 10(6); 337-44. ©2017 AACR.

Interleukin 21 - its potential role in the therapy of B-cell lymphomas.

Interleukin-21 (IL-21), a member of IL-2 cytokine family, has pleotropic biological effects on lymphoid and myeloid cells. During the past 15 years, since the discovery of IL-21, great advances have been made regarding its biological activity and the mechanisms controlling IL-21-mediated cellular responses, especially in hematological malignancies. Preclinical studies have shown that IL-21R is expressed on healthy and neoplastic B-cells and exogenous IL-21 can induce direct apoptosis of IL-21R expressing B-cell non-Hodgkin lymphomas (NHL), making it a potentially attractive anti-lymphoma therapy. However, in some hematological malignancies such as multiple myeloma, Hodgkin lymphoma and Burkitt lymphoma, IL-21 can induce proliferation of neoplastic B-cells. In NHL, the underlying mechanism of cell death was found to be different between the various subtypes, including activation of different JAK/STAT signal transduction pathways or other factors. Immunomodulatory effects of IL-21 have also been reported to contribute to its anti-tumor effects as described by earlier studies in solid tumors and B-cell associated malignancies. These effects are predominantly mediated by IL-21's ability to activate cytolytic activities by NK-cells and CD4+/CD8+ T-cells. In this review, we provide an overview of IL-21's effects in NHL, results from clinical trials utilizing IL-21, and propose how IL-21 can be therapeutically exploited for treating these lymphomas.

Immunomodulatory properties of medicinal mushrooms: differential effects of water and ethanol extracts on NK cell-mediated cytotoxicity.

Medicinal mushrooms have been used for centuries in Asian countries owing to their beneficial effects on health and longevity. Previous studies have reported that a single medicinal mushroom may produce both stimulatory and inhibitory effects on immune cells, depending on conditions, but the factors responsible for this apparent dichotomy remain obscure. We show here that water and ethanol extracts of cultured mycelium from various species (Agaricus blazei Murrill, Antrodia cinnamomea, Ganoderma lucidum and Hirsutella sinensis) produce opposite effects on NK cells. Water extracts enhance NK cell cytotoxic activity against cancer cells, whereas ethanol extracts inhibit cytotoxicity. Water extracts stimulate the expression and production of cytolytic proteins (perforin and granulysin) and NKG2D/NCR cell surface receptors, and activate intracellular signaling kinases (ERK, JNK and p38). In contrast, ethanol extracts inhibit expression of cytolytic and cell surface receptors. Our results suggest that the mode of extraction of medicinal mushrooms may determine the nature of the immunomodulatory effects produced on immune cells, presumably owing to the differential solubility of stimulatory and inhibitory mediators. These findings have important implications for the preparation of medicinal mushrooms to prevent and treat human diseases.

Effects of Rhodomyrtus tomentosa Extract on Killing Activity of Human Neutrophils and Membrane Integrity of Enterohaemorrhagic Escherichia coli O157:H7.

Enterohaemorrhagic Escherichia coli (E. coli) O157:H7 is one of the most virulent causative agents of foodborne disease. Use of antibiotics for the treatment against E. coli O157:H7 infection leads to hemolytic uremic syndrome. The present study evaluated the potential of ethanolic leaf extract of a medicinal plant, Rhodomyrtus tomentosa in enhancing the killing activity of human neutrophils against E. coli O157:H7. In addition, the effects of the extract on membrane permeability of the organisms were studied. In the killing assay, percentage survival of the bacterial cells after being exposed to human neutrophils in the presence of various concentrations of the extract were determined. At 45 min, percentage survival of E. coli O157:H7 and E. coli ATCC 25922 after treated with neutrophils in the presence of the extract at 125-250 µg/mL was 58.48%-50.28% and 69.13%-35.35%, respectively. Furthermore, upon treatment with R. tomentosa at 250 µg/mL uptake of crystal violet by E. coli O157:H7 and E. coli ATCC 25922 was increased to 40.07% and 36.16%, respectively. Therefore, it is suggested that the extract exhibited dual effects as immunostimulant and membrane permeabilizing agent perhaps resulted in enhancing the killing activity of neutrophils against the organisms.

Anti-TNF-α and anti-arthritic effect of patuletin: A rare flavonoid from Tagetes patula.

Rheumatoid arthritis (RA) poses a serious health problem as a chronic autoimmune joint disease with significant mortality and morbidity. Proinflammatory cytokines TNF-α and IL-1ß, reactive oxygen species (ROS), and activated CD4(+) T-cells play key roles in the progression of arthritis. The aim of the study is to evaluate the in vitro and in vivo immunomodulatory and anti-arthritic effect of flavonoid patuletin, isolated from Tagetes patula. ELISA was applied for quantification of TNF-α and IL-1ß. Intracellular and extracellular ROS production from phagocytes was measured by the chemiluminescence technique. Proliferation of T-cells was observed using a liquid scintillation counter. Cytotoxicity was assessed by a MTT assay. The serological and histological analysis studies were performed using a rodent model of adjuvant-induced arthritis (AIA). Expression of p38 and NF-κB after treatment of compound was observed by western blotting. Patuletin showed potent inhibitory effects on TNF-α in vitro as well as inhibited the production of both cytokines in vivo. It also showed potent suppression of proliferation of T-cells and significantly inhibited the extracellular and intracellular ROS production. Patuletin revealed significant anti-inflammatory and anti-arthritic activities in the rodent model of adjuvant-induced arthritis (AIA). Histologically, it causes mild bone destruction compared to the arthritic control group, thus representing its anti-arthritic potential. Based on these studies, patuletin could be considered as a potential immunosuppressive and anti-arthritic lead candidate.

Lavandula angustifolia Mill. Essential Oil Exerts Antibacterial and Anti-Inflammatory Effect in Macrophage Mediated Immune Response to Staphylococcus aureus.

Different studies described the antibacterial properties of Lavandula angustifolia (Mill.) essential oil and its anti-inflammatory effects. Besides, no data exist on its ability to activate human macrophages during the innate response against Staphylococcus aureus. The discovery of promising regulators of macrophage-mediated inflammatory response, without side effects, could be useful for the prevention of, or as therapeutic remedy for, various inflammation-mediated diseases. This study investigated, by transcriptional analysis, how a L. angustifolia essential oil treatment influences the macrophage response to Staphylococcus aureus infection. The results showed that the treatment increases the phagocytic rate and stimulates the containment of intracellular bacterial replication by macrophages. Our data showed that this stimulation is coupled with expression of genes involved in reactive oxygen species production (i.e., CYBB and NCF4). Moreover, the essential oil treatment balanced the inflammatory signaling induced by S. aureus by repressing the principal pro-inflammatory cytokines and their receptors and inducing the heme oxygenase-1 gene transcription. These data showed that the L. angustifolia essential oil can stimulate the human innate macrophage response to a bacterium which is responsible for one of the most important nosocomial infection and might suggest the potential development of this plant extract as an anti-inflammatory and immune regulatory coadjutant drug.