YAC-1 Tumor Cells Research Articles

Crosstalk between NFAT and NFκB Signaling Regulates NK Cell Immunosurveillance

One of the major transcriptional regulators in lymphoid cells is NFAT (Nuclear Factor of Activated T Cells), controlling lymphocyte development and activity. The role of NFAT signaling is well defined in T cells, the cytotoxic lymphocytes of the adaptive immunity. However, surprisingly little is known regarding the relevance of this transcription factor family in NK cells as effector cells of the innate immunity. Available data indicate that NFAT activity is dispensable for development of NK cells, whereas effects of the immunosuppressive drugs cyclosporin A and tacrolimus, that inhibit calcineurin and consecutively NFAT, implicate an involvement of the NFAT family in NK cell function. We here employed different genetic mouse models and functional analyses to unravel the role of NFAT1 (NFATc2) and NFAT2 (NFATc1) in NK cell reactivity. In vitro knockout (KO) of NFAT1 (NFATc2) or NFAT2 (NFATc1) enhanced NK cell degranulation and resulted in increased production of granzyme B and perforin upon stimulation of activating receptors like NK1.1 or Nkp46 or upon co-culture with different leukemia and solid tumor cells. In line, cytotoxicity assays revealed increased lysis of YAC-1 and B16F10 tumor cells by both NFAT1- and NFAT2-deficient NK cells as compared to wildtype (WT) controls. The inhibitory effect of NFAT transcription factors on NK cell effector function could also be confirmed in vivo by employing WT and NFAT KO animals in syngeneic B16F10 melanoma, RMA-S lymphoma and LL/2-luc lung carcinoma models, which revealed a significantly reduced tumor burden in NFAT1 and NFAT2 KO mice. Furthermore, we detected higher numbers of tumor infiltrating NK cells in NFAT KO mice, which suggested improved NK cell migration and homing to the tumor side. Comparative analyses with single NFAT as well as NFAT1+NFAT2 double KO and WT animals further confirmed the inhibitory effect of NFAT1 and NFAT2 and pointed to additive effects of NFAT1 and NFAT2 in NK cell tumor immunosurveillance. Proteomics analysis of NK cells from the different NFAT KO strains revealed altered expression of molecules which are regulating chemokine receptor expression as well as a decreased expression of NFκB inhibitory molecules, like NFKBIB (NFKB inhibitor beta) Commd6 (COMM domain containing 6). NFκB signaling was shown to regulate granzyme B and perforin synthesis and expression. Thus, we suggest that NFAT1 and NFAT2 inhibit NFκB signaling in NK cells, and NFAT1 and NFAT2 KO reinforce NFκB induced granzyme B and perforin expression and thereby increase NK cell cytotoxicity. Taken together, our results identify NFAT as a negative regulator of NK cell function. In addition, we provide the first evidence for a direct functional involvement of NFAT1 and NFAT2 in NK cell antitumor reactivity by regulation tumor infiltration and cytotoxicity of NK cell.

Red Ginseng Extract and γ-Aminobutyric Acid Synergistically Enhance Immunity Against Cancer Cells and Antitumor Metastasis Activity in Mice.

The effects of combined administration of red ginseng (RG) extracts and gamma-aminobutyric acid (GABA) on immunostimulatory activity and tumor metastasis inhibition were investigated in mice. For the immunostimulatory activity, splenocyte proliferation, natural killer (NK) cell activity, including the production of granzyme B (GrB) and interferon gamma (IFN-γ), and serum level of cytokine such as IFN-γ, interleukin (IL)-17, and IL-21 were assessed. Peyer's patch cells obtained from mice administered with RG+GABA were cultured, and the cytokine level in the culture supernatant and bone marrow (BM) cell proliferation activity were examined. The proliferative activity of splenocytes was significantly higher in the RG-GABA treatment group than in RG or GABA alone (P < .05). In the experimental tumor metastasis model, oral administration of RG+GABA showed a higher antitumor metastatic effect compared to that of RG or GABA alone. Oral administration of RG+GABA significantly augmented NK cell-mediated cytotoxicity against YAC-1 tumor cells. In addition, the production of GrB and IFN-γ was stimulated in the culture supernatant of NK cells and YAC-1 cells. Serum concentrations of IFN-γ, IL-17, and IL-21 in mice with RG+GABA were significantly higher compared to the corresponding blood levels in mice administered with RG or GABA alone. The RG+GABA group showed significant BM cell proliferation and increased production of IL-6 and granulocyte-macrophage colony-stimulating factor compared to that in the monotherapy groups. Therefore, RG may have a synergistic effect with GABA for enhancing the host defense system such as BM proliferation and NK cell activity in a tumor metastasis model.

Abstract 2104: NK cell immunosurveillance of tumors is regulated by NFAT1 and NFAT2

Abstract NK cells are lymphoid components of innate immunity and play an important role in tumor immunosurveillance. One of the major transcriptional regulators in lymphoid cells is NFAT (Nuclear Factor of Activated T cells), controlling lymphocyte development and activity. However, while the role of NFAT signaling is well defined in T cells, the cytotoxic lymphocytes of adaptive immunity, surprisingly little is known regarding the relevance of this transcription factor family in NK cells as effector cells of innate immunity. Available data indicate that NFAT activity is dispensable for development of NK cells. However, several lines of evidence including reports on the effects of the immunosuppressive drugs cyclosporin A and tacrolimus, which mediate their effects through inhibition of calcineurin and consecutively NFAT, implicate an involvement of the NFAT family in NK cell function. We here employed different genetic mouse models to unravel the role of NFAT1 (NFATc2) and NFAT2 (NFATc1) in NK cell reactivity. When NK cells with knockout (KO) of NFAT1 or NFAT2 compared to NK cells of control mice (WT) were employed in in vitro analyses, lack of either NFAT was found to surprisingly result in enhanced NK cell degranulation as well as increased production of granzyme B and perforin upon stimulation of activating receptors like NK1.1 or Nkp46 or upon co-culture with different leukemia and solid tumor tumor cells. In line, cytotoxicity assays revealed increased lysis of YAC-1 and B16F10 tumor cells by both NFAT1- and NFAT2-deficient NK cells as compared to WT controls. The inhibitory effect of NFAT transcription factors on NK cell effector function could also be confirmed in vivo by employing WT and NFAT KO animals in syngeneic B16F10 melanoma and RMA-S flank tumor models, which revealed a significantly reduced tumor burden in NFAT1 and NFAT2 KO mice. Comparative analyses with single NFAT as well as NFAT1+NFAT2 double KO and WT animals further confirmed the inhibitory effect of NFAT1 and NFAT2 and pointed to additive effects of NFAT1 and NFAT2 in NK cell tumor immunosurveillance. Taken together, our results provide the first evidence for the direct functional involvement of NFAT1 and NFAT2 in NK cell antitumor reactivity and, in contrast to T and B cells, identify NFAT as a negative regulator of NK cell function. Citation Format: Melanie Märklin, Samuel Holzmayer, Kübra Kaban, Martin R. Müller, Helmut R. Salih. NK cell immunosurveillance of tumors is regulated by NFAT1 and NFAT2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2104.

Egg Yolk Protein Water Extracts Modulate the Immune Response in BALB/c Mice with Immune Dysfunction Caused by Forced Swimming.

The objective of this study was to determine the immunomodulatory effects of egg yolk protein–water extract (EYW) on splenocyte proliferation, cytokine secretion, immunoglobulin production, and NK cell cytotoxic activity in BALB/c mice. The forced swimming test (FST) was used to provide a model for suppressing immune regulation. The proliferation of B cells in the EYW supplementation group was significantly increased from the level to which it was reduced by the FST (from 40.9% to 81.8%, p < 0.05). EYW supplementation affected cytokine secretion of splenocytes. Levels of interleukin (IL)-2 and IL-10—as Th1 and Th2 cytokines, respectively—were decreased after the FST. However, EYW supplementation showed that secretion levels of these cytokines were significantly increased to pre-FST levels (p < 0.05). The production of immunoglobulins (IgA and IgG) was increased abnormally after the FST, whereas EYW supplementation significantly decreased it to pre-FST levels (p < 0.05). EYW supplementation also improved NK cell cytotoxic activity against YAC-1 tumor cells compared to the PC group (p < 0.05). These data suggest that EYW has potential as an immunomodulatory agent in the food and/or pharmaceutical industries.

Constitutive activation of WASp leads to abnormal cytotoxic cells with increased granzyme B and degranulation response to target cells.

X-linked neutropenia (XLN) is caused by gain-of-function mutations in the actin regulator Wiskott-Aldrich Syndrome protein (WASp). XLN patients have reduced numbers of cytotoxic cells in peripheral blood; however, their capacity to kill tumor cells remains to be determined. Here, we examined NK and T cells from 2 patients with XLN harboring the activating WASpL270P mutation. XLN patient NK and T cells had increased granzyme B content and elevated degranulation and IFN-γ production when compared with healthy control cells. Murine WASpL272P NK and T cells formed stable synapses with YAC-1 tumor cells and anti-CD3/CD28–coated beads, respectively. WASpL272P mouse T cells had normal degranulation and cytokine response whereas WASpL272P NK cells showed an enhanced response. Imaging experiments revealed that while WASpL272P CD8+ T cells had increased accumulation of actin upon TCR activation, WASpL272P NK cells had normal actin accumulation at lytic synapses triggered through NKp46 signaling but had impaired response to lymphocyte function associated antigen-1 engagement. When compared with WT mice, WASpL272P mice showed reduced growth of B16 melanoma and increased capacity to reject MHC class I–deficient cells. Together, our data suggest that cytotoxic cells with constitutively active WASp have an increased capacity to respond to and kill tumor cells.

A High-Salt Diet Disturbs the Development and Function of Natural Killer Cells in Mice.

A high-salt diet (HSD) is common worldwide and can lead to cardiovascular disease, chronic inflammation, and autoimmune diseases. Moreover, increasing evidence shows that HSD is closely related to a variety of immune diseases. Natural killer (NK) cells are important innate immune cells that directly kill their targets via degranulation and secretion of interferon gamma (IFN-γ). NK cells play a vital role in resisting viruses and preventing the malignant transformation of cells; however, whether HSD affects the development and function of NK cells has not yet been elucidated. Therefore, the purpose of the present study was to understand the effects of HSD on the development and function of NK cells, in addition to investigating the underlying molecular mechanism. Our results show that the number of NK cells in the spleen and lungs of HSD-fed mice was significantly reduced, which may be due to the inhibition of NK cell proliferation. Further, the development of NK cells in mice was evaluated, and it was found that HSD reduced the effective NK cell subset (CD27+CD11b−). Moreover, it was also found that the ability of NK cells to secrete CD107a and IFN-γ in HSD-fed mice was decreased following stimulation with RMA-S and YAC-1 tumor cells. Finally, the underlying molecular mechanism was evaluated, and it was found that HSD increased the production of reactive oxygen species (ROS) by NK cells, while the expression of CD122 was decreased, suggesting that HSD downregulates CD122 expression in NK cells via ROS signaling, thereby reducing the responsiveness to IL-15 and ultimately inhibiting NK cell function. The present research discovered a novel mechanism by which HSD inhibits the function of NK cells, providing an alternative avenue for the treatment of immune diseases caused by HSD.

Phase 1 Trial of Prophylactic Donor-Derived IL-2 Activated NK Cell Infusion after Allogeneic Hematopoietic Stem Cell Transplantation from a Matched Sibling Donor

Background: Donor lymphocyte infusion (DLI) is commonly used to enhance T-cell mediated graft-versus-tumor (GVT) effect after allogeneic stem cell transplantation (allo-HSCT). It was showed that prophylactic DLI can produce promising outcome but is also associated with a high rate of graft-versus-host disease (GVHD), limiting its use as maintenance treatment. By contrast with T cells, there are clinical and biological evidences that NK cells can provide strong GVT effect without inducing GVHD, making post allo-HSCT NK cell-based immunotherapy appealing. Thus, we designed a phase 1 clinical trial evaluating the safety of donor-derived ex vivo IL-2 activated NK cells (DLI-NK) as maintenance immunotherapy after allo-HSCT (DLI-NK: NCT01853358). Methods: We included patients who were in morphologic remission of hematological cancers 2 months after allo-HSCT from a matched sibling donor after reduced intensity conditioning regimen, without history of acute GVHD. NK cells were purified from the donor (CD3 depletion / CD56 positive selection) and cultured ex vivo during 7 days with IL-2 before prior to infusion in patients between day+60 and day+90 after allo-HSCT. The study included a first phase of dose escalation (3+3 method) to determine the maximal tolerated dose (MTD) using 3 dose levels: 1.0 (dose 1), 5.0 (dose 2), 5.0-50 (dose 3) million cells/kg. A second phase allowed the treatment of subsequent patients to reach a total amount of 10 patients treated at the MTD. As ancillary studies, we performed: 1) preclinical evaluation of donor-derived IL-2 stimulated NK cell therapy in mice; 2) phenotypical and functional characterization of the NK cell therapy product before and after 7-day IL-2 stimulation, and 3) longitudinal immunomonitoring of NK cell phenotype in patients. Results: We treated 16 patients without observing any dose-limiting toxicity (grade 3-4 adverse events related to DLI-NK within 30 days after infusion). During the follow up, 4 patients developed chronic GVHD (1 mild, 2 moderate and 1 severe forms). All of them were in complete remission of GVHD without immunosuppressive treatment at last contact. We observed one non-relapse death due to idiopathic pneumonia. From the four patients who relapsed (3 AML and 1 CLL), 2 died from their disease and 2 were in complete response after salvage therapy. At last follow up, 11 out of 16 treated patients were alive in CR without GVHD features and immunosuppressive treatment. Ancillary studies showed that IL-2 stimulated allogeneic NK cells did not induce GVHD and displayed strong antitumor effect in mice against YAC-1 tumor cells, supporting the efficacy of this approach. In human, we observed that IL-2-activated NK cells expressed high levels of activating receptors (DNAM-1, NKG2A, NKp46, NKp30) and of the inhibitory receptor NKG2A, and exhibited increased degranulation and cytokine production when challenged by K562 tumor cells, as compared to freshly isolated NK cells. Immunomonitoring of patient NK cells after DLI-NK do not reveal significant phenotypic or functional dysfunction, with a pattern of NK cell recovery that is similar to what was previously observed after allo-HSCT. Conclusion: We showed that prophylactic DLI-NK after matched sibling donor allo-HSCT was feasible and safe. We observed low incidence of GVHD as compared to previous reports of conventional prophylactic DLI, reinforcing the rationale of NK cell role in GVHD prevention. In addition, promising disease-free survival supports the design of future clinical trials evaluating the efficacy of DLI-NK as disease relapse prevention. Disclosures Harbi: Sanofi: Honoraria. Olive:Imcheck Therapeutics: Other: Co-Founder &amp; shareholder; Emergence Therapeutics: Other: Co-Founder &amp; shareholder; Alderaan Biotechnology: Other: Co-Founder &amp; shareholder. Blaise:Jazz Pharmaceuticals: Honoraria.

Artemisinin sensitizes tumor cells to NK cell-mediated cytolysis

The antimalarial drug Artemisinin has been reported to possess direct anti-tumor effects on various types of tumor cells. However, its anti-tumor potential has not been fully revealed, and its effects on tumor susceptibility to immune surveillance by the host are still unknown. Natural killer (NK) cells are the first line in tumor surveillance by the host, and have been recognized as a promising target for tumor immunotherapy. Here, we reported that Artemisinin sensitized tumor cells to NK cell cytolysis. Both human K562 and Raji tumor cells, and mouse YAC-1 tumor cells were more susceptible to human or mouse NK cell cytolysis in vitro after Artemisinin pretreatment. Conjugation formation between tumor cells and NK cells was increased after pretreatment with Artemisinin. Such effects on tumor cells by Artemisinin might not be the results of tumor recognition by NK cells, since major ligands of NK cell surface receptors were not affected. Mechanistically, although Artemisinin didn’t induce tumor cell apoptosis, Artemisinin enriched apoptosis-related gene sets in these tumor cells, which might predispose tumor cells to apoptosis upon NK cell cytolysis. Moreover, NK cell numbers, percentages, maturation and functions were preserved in the presence of Artemisinin in vitro, suggesting that Artemisinin displays detrimental effects only on tumor cells but not on immune cells. These data reveal a novel anti-tumor mechanism of Artemisinin and demonstrate that Artemisinin could be a promising drug candidate for cancer treatment.

Sesamum indicum (sesame) enhances NK anti-cancer activity, modulates Th1/Th2 balance, and suppresses macrophage inflammatory response

Objective: To evaluate the potential immunomodulatory effects of an aqueous extract of Sesamum indicum seeds with regard to splenocyte proliferation, Th1/Th2 balance, macrophage function, and the cytotoxic activity of natural killer (NK) cells. Methods: Splenocyte proliferation was measured by [3H]- thymidine incorporation. Griess assay was performed to evaluate the production of nitric oxide by macrophages. The levels of cytokines secreted by splenocytes and macrophages were measured by ELISA. JAM assay was performed to examine the cytotoxic activity of NK cells against YAC-1 tumor cells. Results: Sesamum indicum significantly enhanced splenocyte proliferation in a dose-dependent manner. Sesamum indicum also increased and suppressed the secretion of Th1 and Th2 cytokines, respectively, by splenocytes. The secretion of key pro-inflammatory mediators (IL-6, TNFα, and nitric oxide) by primary macrophages was significantly inhibited by Sesamum indicum. Moreover, Sesamum indicum increased the cytotoxic activity of NK cells against YAC-1 tumor cells. Conclusions: Sesamum indicum shows potent immunomodulatory, anti-inflammatory, and anti-cancer effects. Constituents of Sesamum indicum may be used as effective therapeutic agents in regulating immune reactions implicated in various infectious and non- infectious conditions including cancer.

Rhamnogalacturonan-I-Type Polysaccharide Purified from Broccoli Exerts Anti-Metastatic Activities Via Innate Immune Cell Activation.

To examine the anti-metastatic activities of polysaccharides in broccoli, purified polysaccharides (BCE-I, -II, and -III) were isolated by fractionation of broccoli enzyme extracts and subsequent ethanol precipitation. BCE-I mainly consisted of galactose and arabinose, whereas BCE-II mainly consisted of galacturonic acid and rhamnose, and BCE-III mainly consisted of rhamnose and galactose. Of the three fractions, stimulation of murine peritoneal macrophages by BCE-I showed the greatest enhancement of tumor necrosis factor-α, interleukin (IL)-12, and IL-6 secretion. In addition, intravenous (i.v.) administration of BCE-I enhanced the lethal activity of natural killer (NK) cells on YAC-1 tumor cells significantly and dose-dependently in an ex vivo experiment of NK cell activity. In an experimental model using lung metastasis of Colon26-M3.1 carcinoma cells, prophylactic i.v. and oral administration of BCE-I significantly and dose-dependently inhibited lung metastatic activity. Furthermore, the inhibitory activity of BCE-1 on lung metastasis partially disappeared when NK cell function was removed through treatment of rabbit anti-asialo GM1. These results indicated that BCE-I has potent antitumor metastatic activity, and that its anti-metastatic activity has relevance to the stimulation of NK and other immune cells.

Immunostimulating and Antimetastatic Effects of Polysaccharides Purified from Ginseng Berry.

Ginseng root has been used in traditional oriental medicine for the enhancement of immune system function. The immunostimulatory effects of ginseng berry polysaccharides, however, remain unclear. Effects of polysaccharides from ginseng berry on the activation of natural killer (NK) cells and inhibition of tumors are reported. A crude polysaccharide was isolated from ginseng berry as a ginseng berry polysaccharide portion (GBPP) and was further fractionated using gel filtration chromatography to obtain the three polysaccharide fractions GBPP-I, -II and -III. GBPP-I consisted of mainly galactose (46.9%) and arabinose (27.5%). GBPP-I showed a high dose-dependent anticomplementary activity. Stimulation of murine peritoneal macrophages by GBPP-I showed the greatest enhancement of interleukin (IL)-6 and IL-12 and tumor necrosis factor (TNF)- production. In addition, an ex vivo assay of natural killer (NK) cell activity showed that oral ( administration of GBPP-I significantly increased NK cell cytotoxicity in YAC-1 tumor cells and production of granzyme B. Prophylactic intravenous ( and administration of GBPP-I significantly and dose-dependently inhibited lung metastatic activity in B16BL6 melanoma cells. Depletion of NK cells after injection of rabbit anti-asialo GM1 partially abolished the inhibitory effect of GBPP-I on lung metastasis, indicating that NK cells play an important role in anticancer effects. GBPP-I exerts a strong immune-enhancing activity and can prevent cancer metastasis through activation of NK cells and other immune-related cells.

Tyndallized Lactobacillus plantarum HY7712 Restores Whole-Body γ-Irradiation-Impaired Th Cell Differentiation in Mice.

In the present study, we investigated the effect of tyndallized HY7712 (tHY7712) on the expression of Th cell specific transcription factors and cytokines in whole-body γ-irradiated mice. Oral administration of tHY7712 strongly recovered the γ-irradiation-suppressed expression of helper T (Th) cell- and regulatory T cell-related transcription factors and cytokines, such as T-bet, Foxp3, IFN-γ, TNF-α, and IL-10, and suppressed Th2 cell-associated transcription factor and cytokine GATA3 and IL-5, respectively. Furthermore, compared with the control, tHY7712 treatment also restored γ-irradiation-impaired natural killer and cytotoxic T cell activities against YAC-1 tumor cells to 97.8% and 98.6%, respectively.

The Abl-1 Kinase is Dispensable for NK Cell Inhibitory Signalling and is not Involved in Murine NK Cell Education.

Natural killer (NK) cell responsiveness in the mouse is determined in an education process guided by inhibitory Ly49 and NKG2A receptors binding to MHC class I molecules. It has been proposed that inhibitory signalling in human NK cells involves Abl-1 (c-Abl)-mediated phosphorylation of Crk, lowering NK cell function via disruption of a signalling complex including C3G and c-Cbl, suggesting that NK cell education might involve c-Abl. Mice deficient in c-Abl expression specifically in murine NK cells displayed normal inhibitory and activating receptor repertoires. Furthermore, c-Abl-deficient NK cells fluxed Ca2+ normally after triggering of ITAM receptors, killed YAC-1 tumour cells efficiently and showed normal, or even slightly elevated, capacity to produce IFN-γ after activating receptor stimulation. Consistent with these results, c-Abl deficiency in NK cells did not affect NK cell inhibition via the receptors Ly49G2, Ly49A and NKG2A. We conclude that signalling downstream of murine inhibitory receptors does not involve c-Abl and that c-Abl plays no major role in NK cell education in the mouse.

Polysaccharides from ginseng leaves inhibit tumor metastasis via macrophage and NK cell activation

In this study, we purified the polysaccharide fraction (GS-P) from the leaves of Panax ginseng C.A. Meyer and analyzed its monosaccharide composition and antitumor and antimetastatic activity in vitro and in vivo. GS-P is a 10.2kDa pectic polysaccharide consisting of 15 different monosaccharides. GS-P treatment significantly inhibited metastasis in mice, in a dose-dependent manner. GS-P was not cytotoxic to colon 26-M3.1 cells and increased mouse splenocyte proliferation. Secretion of tumor necrosis factor (TNF)-α and interleukin (IL)-12 was enhanced in the peritoneal exudate macrophages (PEMs) of GS-P-treated mice. Moreover, PEMs obtained from GS-P-treated mice showed significantly higher tumoricidal activity against colon 26-M3.1 cells, and splenocytes from GS-P-treated mice significantly enhanced NK cell cytotoxicity against YAC-1 tumor cells. Pretreatment with anti-asialo GM1 (an antibody for NK cell depletion) partly suppressed the inhibitory effects of GS-P on lung metastasis. These data suggest that GS-P exhibits antimetastatic activity by promoting the activation of macrophages and NK cells.

Antitumor and antimetastatic activities of rhamnogalacturonan-II-type polysaccharide isolated from mature leaves of green tea via activation of macrophages and natural killer cells

To investigate the antitumor and antimetastatic polysaccharide from the mature leaves of green tea, GTE-II was purified using size exclusion chromatography. GTE-II consisted of 15 different sugars including rarely observed sugars such as 2-O-methyl-fucose, 2-O-methyl-xylose, apiose, aceric acid, 3-deoxy-d-manno-2-octulosonic acid, and 3-deoxy-d-lyxo-2-heptulosaric acid, which were characteristics of pectic polysaccharide rhamnogalacturonan-II. Treatment of peritoneal macrophages with GTE-II not only increased interleukin (IL)-6 and IL-12 production, but also had significantly increased tumoricidal activity against Yac-1 tumor cells than those obtained from untreated mice. In an assay of natural killer (NK) cell activity, intravenous administration of GTE-II significantly stimulated NK cytotoxicity against Yac-1 tumor cells. Furthermore, the depletion of NK cells by injection of rabbit anti-asialo GM1 serum eliminated the inhibitory effect of GTE-II on B16BL6 melanoma cells. These data suggest that GTE-II inhibits tumor metastasis, and its antitumor effect is associated with activation of macrophages and NK cells.

Effects of dietary mixture of garlic (Allium sativum), coriander (Coriandrum sativum) and probiotics on immune responses and caecal counts in young laying hens

This study was conducted to evaluate the effects of a combined mixture of phytogenic extracts (garlic and coriander) and probiotics on growth performance and immune responses in laying hens based on the results of invitro studies to screen for immunomodulatory potency of each ingredient. Several parameters of immunomodulatory potency were estimated using lamina propria leucocytes (LPLs) isolated from rat intestinal mucosa tissue. Results show that the combined mixture enhanced LPLs proliferation, increased LPL-mediated cytotoxicity against YAC-1 tumour cells, and decreased lipopolysaccharide (LPS)-induced cytokine production including tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in LPLs. For invivo study, laying hens (n=50/each diet group) were fed with control diet, a diet containing antibiotics (0.01% per kg feed) or the combined mixture (0.02% per kg feed) for 21days. The dietary combined mixture improved egg production (p<0.05) but not growth performance and carcass traits. Interestingly, the patterns of suppressing plasma IFN-γ productions during inflammation by LPS injection and decreasing caecal E.coli counts in the combined mixture group were comparable to those in the antibiotics group. Taken together, our results suggested that the 0.02% of combined mixture of phytogenic extracts and probiotics as ingredients has potential immunomodulatory effects in laying hens.

Uptake of poly-dispersed single-walled carbon nanotubes and decline of functions in mouse NK cells undergoing activation

The interaction of poly-dispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNT) with NK cells undergoing activation was examined. Exposure to AF-SWCNT during NK activation in vitro by interleukin (IL)-2, and in vivo by Poly(I:C) significantly lowered cytotoxic activity generated against YAC-1 tumor cells. Recoveries of spleen NK1.1+ cells as well as the activated subset of NK cells (NK1.1+CD69+ cells) were significantly reduced by the AF-SWCNT exposure. The proportion of apoptotic NK cells (NK1.1+ phosphatidylserine+) in the spleen cell preparations activated in vitro was also significantly elevated. Expression levels of CD107a [for assessing NK cell degranulation] as well as of FasL marker [mediating non-secretory pathway of NK cell killing] were significantly lower in cells exposed to AF-SWCNT during the activation phase. Intracellular levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α in the cells were also significantly reduced. Fluorescent AF-SWCNT (FAF-SWCNT) were internalized by the NK cells and uptake was significantly greater in activated cells. Confocal microscopy indicated the internalized FAF-SWCNT were localized to the cytoplasm of the NK cells. These results indicated that AF-SWCNT were internalized by NK cells and caused a general down-regulation of a variety of parameters associated with NK cell cytotoxicity and other cellular functions.

Antitumor metastasis activity of pectic polysaccharide purified from the peels of Korean Citrus Hallabong

A polysaccharide fraction, HBE-III, was successfully purified in a high yield (40.4%) from its crude polysaccharide (HBE-0) which was prepared from pectinase hydrolysates of the peels of the Korean Citrus Hallabong. In experimental lung metastasis studies of Colon 26-M3.1 carcinoma cells, prophylactic administration of HBE-III significantly inhibited lung metastasis in a dose-dependent manner. In an in vitro cytotoxicity analysis, HBE-III (up to 1000μg/mL) did not affect the growth of Colon 26-M3.1 cells and normal cells. HBE-III enhanced production of IL-6 and IL-12 by murine peritoneal macrophages. In an assay for natural killer (NK) cell activity, HBE-III (1000μg/mouse, i.v.) significantly augmented NK cytotoxicity against Yac-1 tumor cells. The depletion of NK cells by injection of mouse anti-asialo GM1 serum abolished the inhibitory effect of HBE-III on lung metastasis of Colon 26-M3.1 cells. These data suggest that HBE-III may inhibit tumor metastasis via activation of macrophages and NK cells.

Lactobacillus plantarum HY7712 Protects Against the Impairment of NK-Cell Activity Caused by Whole-Body ��-Irradiation in Mice

While searching for lactic acid bacteria that can restore aging-impaired immune responses, we isolated the Toll-like receptor (TLR) 2/NF-kappaB-activating strain Lactobacillus plantarum HY7712 from kimchi and investigated its immunomodulating effect in whole-body γ-irradiated mice. Exposure to HY7712 strongly activated NF-kappaB signaling in RAW264.7 cells, but inhibited lipopolysaccharide-stimulated NF-kappaB activation. Moreover, HY7712 protected against the downregulation of interferon (IFN)-γ and upregulation of interleukin (IL)-13 caused by γ-irradiation in mice. In mice, γ-irradiation impaired NK-cell activity against YAC-1 tumor cells, but following HY7712 exposure, the activity of NK cells was restored to 91.5% of the level measured in control mice (p < 0.05). These findings suggest that HY7712 activates the TLR2/NF-kappaB signaling pathway and protects against the impairment of NK-cell activity caused by γ-irradiation or aging.

땃두릅(Oplopanax elatus Nakai) 추출물의 면역자극 활성 및 항암 증진 효과

The present study is designed to explore an anti-tumor activity on crude extracts of Oplopanax elatus. Water extractions of Oplopanax elatus were performed at <TEX>$100^{\circ}C$</TEX>(OeE-100). OeE-100 doses up to <TEX>$62.5{\mu}g/m{\ell}$</TEX> had no cytotoxicity on the tumor cell lines in vitro. In experimental lung metastasis of colon26-M3.1 carcinoma or B16-BL6 melanoma, the prophylactic intravenous (<TEX>$4{\sim}100{\mu}g/mouse$</TEX>) or oral (2 mg/mouse) administration of OeE-100 significantly inhibited tumor metastasis as compared with tumor controls. Peritoneal macrophages stimulated with OeE-100 produced various cytokines such as TNF-<TEX>${\alpha}$</TEX>, IL-6 and IL-12. In an analysis of NK-cell activities, i.v. administration of OeE-100 (<TEX>$10{\sim}100{\mu}g/mouse$</TEX>) significantly augmented the cytotoxicity to YAC-1 tumor cells. Vaccination of mice with boiling-treated tumor cells (BT-vaccine) in combination with OeE-100 (<TEX>$100{\mu}g/mouse$</TEX>) showed higher inhibitions in tumor metastasis when compared with the mice of BT-vaccine treatment. In addition, the splenocytes from OeE-100 admixed BT-vaccine immunized mice secreted a higher concentration of Th1 type cytokine such as IFN-<TEX>${\gamma}$</TEX>. These results suggested that the OeE-100 stimulated immune system and was a good candidate adjuvant of anti-tumor immune responses.