The Antiproliferation Effect of Berbamine on K562 Resistant Cells by Inhibiting NF‐κB Pathway

Imatinib mesylate is effective against Ph chromosome‐positive leukemia; however, resistance has been reported. High expression of bcr‐abl in mRNA and protein levels, and other alterations were found in patients who experienced imatinib treatment failures and thus it is important to design alternative treatment strategies. The aim of this study was to evaluate the in vitro effect of berbamine, on imatinib‐resistant chronic myelogenous leukemia (CML) K562 (K562‐r) cells, and explore the mechanisms. The growth of K562‐r cells was examined using the 3‐(4,5‐dimethylthiazol‐2yl)‐2,5‐diphenyl‐tetrazolium bromide (MTT) assay. Morphological analysis and DNA agarose electrophoresis were used to detect apoptosis in K562‐r cells, and the extent of the cells in the sub‐G1 cell cycle phase was measured using flow cytometry. The expression levels of BCR‐ABL, phospho‐BCR‐ABL, and nuclear factor κB (NF‐κB), IκBα, phospho‐IκBα, IκB kinases α(IKKα), and Survivin were determined by Western blot. bcr‐abl mRNA expression was determined by RT‐PCR. MTT assays indicated that berbamine significantly inhibited the proliferation of K562‐r cells. Cells with characteristics of apoptosis were confirmed by morphology examination and DNA agarose electrophoresis and percentage of apoptosis were increased after treatment with berbamine. The results also showed that berbamine was able to down‐regulate BCR‐ABL and phospho‐BCR‐ABL proteins by affecting bcr‐abl mRNA expression and decrease expression of nuclear NF‐κB, phospho‐IκBα, IKKα, and Survivin. Collectively, berbamine could inhibit the proliferation of K562‐r cells and induce apoptosis. The mechanisms may be related at least in part, to inhibit BCR‐ABL and its downstream NF‐κB signaling. Berbamine may provide an alternative candidate for the treatment of patients with CML resistant to imatinib therapy. Anat Rec, 292:945–950, 2009. © 2009 Wiley‐Liss, Inc.     

Sensitization of human breast cancer cells to natural killer cell‐mediated cytotoxicity by proteasome inhibition

The proteasome inhibitor, bortezomib, has direct anti‐tumour effects and has been demonstrated to sensitize tumour cells to tumour necrosis factor‐related apoptosis‐inducing ligand‐mediated apoptosis. Natural killer (NK) cells are effective mediators of anti‐tumour responses, both through cytotoxic granule killing and apoptosis‐inducing pathways. We therefore investigated if bortezomib sensitized human breast cancer cells to killing by the human NK cell line, NK‐92. Bortezomib was unable to sensitize MDA‐231 breast cancer cells to NK cell‐mediated killing in short‐term in vitro assays. However, bortezomib did cause these cells to up‐regulate apoptosis‐related mRNA as well as death receptors on the cell surface. In a long‐term in vitro tumour outgrowth assay that allows NK cells to use their full repertoire of killing pathways, bortezomib sensitized three breast cancer cell lines to NK cell‐mediated killing, which led to greater anti‐tumour effects than either treatment alone. We then used a xenogeneic mouse model in which CB‐17 SCID mice were injected with human breast cancer cells. This model displayed the effectiveness of NK‐92 cells, but the addition of bortezomib did not increase the survival further or reduce the number of lung metastases in tumour‐bearing mice. However, while bortezomib was highly cytotoxic to NK‐92 cells in vitro, bortezomib treatment in vivo did not decrease NK‐92 function, suggesting that through alternative dosing or timing of bortezomib, greater efficacy may occur from combined therapy. These data demonstrate that combined treatment of human breast cancer with bortezomib and NK cells has the potential to generate superior anti‐tumour responses than either therapy alone.     

重组人可溶性TRAIL的制备及其联合硼替佐米诱导肿瘤细胞的凋亡作用

目的:构建重组人肿瘤坏死因子相关的凋亡诱导配体(tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)原核表达质粒p ET-28a(+)-TRAIL114-281,优化蛋白表达和纯化条件,制备重组人可溶性TRAIL并鉴定其活性。方法:使用CCK-8初步验证TRAIL是否具有抑制肿瘤细胞生长的生物活性;将制备的TRAIL单独或联合50 nmol/L硼替佐米应用于H460细胞(对TRAIL敏感)和K562细胞(对TRAIL抵抗)24 h,流式细胞术检测细胞凋亡率,比色法检测caspase-8、-9、-3的活化程度,Western blot分析细胞中Bax、Bcl-2和c FLIP蛋白的表达。流式细胞术检测硼替佐米处理H460细胞和K562细胞24 h后DR4和DR5的表达量变化。结果:制备了具有生物学活性且性质稳定的重组人可溶性TRAIL,且成功诱导H460和K562细胞凋亡。不同浓度TRAIL处理H460细胞后其凋亡率随着TRAIL浓度升高而显著升高(P0.05),但K562细胞凋亡率并未随着TRAIL浓度明显升高。联合用药组的H460和K562细胞凋亡率均显著高于单独用药组(P0.05),凋亡过程中caspase-8、-9、-3均被活化,药物处理组的Bcl-2和c FLIP表达量均比对照组下降,尤其联合用药组表达量下降最为显著(P0.05),而Bax表达量无明显变化。硼替佐米处理H460和K562细胞后DR4和DR5表达量均上调(P0.05)。结论:硼替佐米能协同TRAIL启动内源性凋亡途径诱导H460和K562细胞凋亡,其可能机制是通过上调死亡受体DR4和DR5的表达量、下调抗凋亡蛋白Bcl-2和c FLIP的表达量来实现的。     

Bortezomib‐induced pro‐inflammatory macrophages as a potential factor limiting anti‐tumour efficacy

Multiple myeloma (MM) is a chronic progressive malignancy of plasma cells. Although treatment with the novel proteasome inhibitor, bortezomib, significantly improves patient survival, some patients fail to respond due to the development of de novo resistance. We have previously shown that cytotoxic drugs can induce pro‐tumorigenic host‐mediated effects which contribute to tumour re‐growth and metastasis, and thus limit anti‐tumour efficacy. However, such effects and their impact on tumour cell aggressiveness have not been investigated using cytostatic agents such as bortezomib. Here we show that plasma from bortezomib‐treated mice significantly increases migration, viability and proliferation of MM cells in vitro, compared to plasma from vehicle treated mice. In vivo, bortezomib induces the mobilization of pro‐angiogenic bone marrow cells. Furthermore, mice treated with bortezomib and subsequently were used as recipients for an injection of MM cells succumb to MM earlier than mice treated with the vehicle. We show that bortezomib promotes pro‐inflammatory macrophages which account for MM cell aggressiveness, an effect which is partially mediated by interleukin‐16. Accordingly, co‐inoculation of MM cells with pro‐inflammatory macrophages from bortezomib‐treated mice accelerates MM disease progression. Taken together, our results suggest that, in addition to the known effective anti‐tumour activity of bortezomib, host‐driven pro‐tumorigenic effects generated in response to treatment can promote MM aggressiveness, and thus may contribute to the overall limited efficacy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

1140919448CD200-dependent suppression of human NK activity by IVIG requires CD200 receptor type 2

Problem: IVIG prepared from plasma of stored human blood can be efficacious in improving pregnancy success in a selected subgroup of patients but RCTs using an IVIG showing inferior suppression of NK activity in vitro have been negative (J Assist Reprod Genet 2006). A significant component of NK suppression by IVIG appears to be due to CD200 released into plasma from PBL during storage at 4C. CD200 receptors (CD200R) are expressed at the fetomaternal interface prior to onset of abortion; CD200R1 mediates direct effects on gamma‐delta T cell development and suppresses alpha‐beta T cell responses in vitro, whereas CD200R2 alters DC so as to facilitate development of alpha‐beta Treg cells. Which receptor(s) mediate NK cell suppression? Methods: Purified human PBL or the CD56⁺ NK cell subset of PBL were used to lyse 51Cr‐labeled K562 cells in vitro. Different IVIG preparations were tested for suppressive ability, and suppression was blocked by either anti‐huCD200 mAb or rabbit anti‐huCD200R1 or R2 antibodies. Results: CD200‐dependent IVIG NK suppressive potency differed among IVIG types (Gammagard>Gamunex>>Gamimmune). CD200‐dependent suppression was blocked by anti‐CD200R antibody able to react with the type 2 receptor. K562 cells did not express receptor, and purified CD56⁺ NK cells were suppressed effectively without the need for non‐NK cells. Conclusions: IVIG may directly express NK cell activity via CD200 binding to CD200R2.     

Dichloromethane fraction of Melissa officinalis induces apoptosis by activation of intrinsic and extrinsic pathways in human leukemia cell lines

Abstract

Various components from medicinal plants are currently used in cancer therapy because of their apoptosis-inducing effects. The present study has aimed to investigate the growth inhibitory and apoptotic effects of Melissa officinalis on tumor cells. We prepared different fractions of this plant to investigate their inhibitory effects on two leukemia cell lines, Jurkat and K562. Fractions with the highest inhibitory effects were examined for induction of apoptosis by the annexin V/propidium iodide assay and cell cycle changes by flow cytometry. Real-time polymerase chain reaction evaluated the changes in expression of apoptosis-related genes. Among different fractions, dichloromethane and n-hexane dose-dependent showed the strongest inhibitory effects on both K562 and Jurkat cells. The dichloromethane fraction significantly induced apoptosis at concentration of 50?µg/ml on Jurkat (85.66?±?4.9%) and K562 cells (65.04?±?0.93%) at 24?h after treatment (p?<?0.002). According to cell cycle analysis, more than 70% of the cells accumulated in the sub-G1 phase when cultured in the presence of the dichloromethane fraction. This fraction up-regulated Fas and Bax mRNA expression as well as the Bax/Bcl-2 ratio according to cell type, showing its effect on the activation of both extrinsic and intrinsic pathways of apoptosis. The expression of apoptosis-related genes did not significantly change following treatment with the n-hexane fraction. These data indicated that the dichloromethane fraction of M. officinalis had the ability to induce apoptosis and change apoptosis-related gene expression in leukemia cells.     

3种反义核酸增加白血病细胞K562对顺铂的敏感性

目的：研究和探索hTERT、bcl-2、c-myc基因的反义核酸对K562细胞顺铂（cisplatin）敏感性影响,以期增强顺铂对白血病疗效。方法：采用MTT法检测3种反义核酸和顺铂对K562细胞抑制率。结果：顺铂20μmol/L对K562细胞抑制率为17.17%±1.36%,顺铂+hTERT反义序列对K562细胞抑制率为25.41%±1.77%,顺铂+bcl-2反义序列对K562细胞抑制率为26.18%±1.43%,顺铂+c-myc反义序列对K562细胞抑制率为28.29%±1.05%。结论：hTERT、bcl-2、c-myc基因的反义核酸能显著提高K562白血病细胞对顺铂的敏感性。     

Issue Information ‐ TOC

The proteasome inhibitor bortezomib has been widely used to treat patients with multiple myeloma (MM). However, some patients show primary or secondary resistance. In recent work published in The Journal of Pathology, Beyar‐Katz et al demonstrate that bortezomib treatment stimulates a host inflammatory response, which in turn promotes MM cell migration, viability, and proliferation. These effects appear to be mediated by pro‐inflammatory M1‐like stromal macrophages partly via secretion of cytokine IL‐16. These unexpected findings imply that the binary M1/M2 definition of macrophages may not accurately describe the complexity and heterogeneity of macrophages associated with MM tumour growth and progression, and further suggest that bortezomib treatment stimulates host‐driven tumour‐promoting activity in addition to its cytotoxic activity, thus leading to potential bortezomib resistance in MM patients. Understanding the underlying mechanisms may identify novel targets to overcome or prevent bortezomib resistance. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

HAMLET a human milk protein-lipid complex induces a pro-inflammatory phenotype of myeloid cells

HAMLET is a protein-lipid complex with a specific and broad bactericidal and tumoricidal activity, that lacks cytotoxic activity against healthy cells. In this study, we show that HAMLET also has general immune-stimulatory effects on primary human monocyte-derived dendritic cells and macrophages (Mo-DC and Mo-M) and murine RAW264.7 macrophages. HAMLET, but not its components alpha-lactalbumin or oleic acid, induces mature CD14^low/–CD83⁺ Mo-DC and M1-like CD14⁺CD86⁺⁺ Mo-M surface phenotypes. Concomitantly, inflammatory mediators, including IL-2, IL-6, IL-10, IL-12 and MIP-1α, were released in the supernatant of HAMLET-stimulated cells, indicating a mainly pro-inflammatory phenotype. The HAMLET-induced phenotype was mediated by calcium, NFκB and p38 MAPK signaling in Mo-DCs and calcium, NFκB and ERK signaling in Mo-M as inhibitors of these pathways almost completely blocked the induction of mature Mo-DCs and M1-like Mo-M. Compared to unstimulated Mo-DCs, HAMLET-stimulated Mo-DCs were more potent in inducing T cell proliferation and HAMLET-stimulated macrophages were more efficient in phagocytosis of Streptococcus pneumoniae in vitro. This indicates a functionally activated phenotype of HAMLET-stimulated DCs and macrophages. Combined, we propose that HAMLET has a two-fold anti-bacterial activity; one inducing direct cytotoxic activity, the other indirectly mediating elimination of bacteria by activation of immune cells of the myeloid lineage.     

Estrogen suppresses heptatic IκB expression during short-term alcohol exposure

Objective

To assess the effects of sex steroids on hepatic inflammatory pathways in short-term chronically ethanol-fed rats.

Methods

Ovariectomized female Wistar rats (8–12?weeks old, n?=?8 per treatment group) were implanted with osmotic pumps releasing 17β-estradiol (20?μg/24?h) or testosterone (25?μg/24?h) and fed liquid diets with or without ethanol (8?% w/v) for two?weeks. Hepatic expression of IκBα/β, TNF-α, and IL-6 mRNA was examined by real-time PCR. Liver (nuclear) NFκB, IκBα and β, IL-6, and IL-6Rα protein expression was examined by enzyme-linked immunosorbent assay (ELISA) or Western blot.

Results

Estrogen alone induced greater steatosis, NFκB translocation, TNF-α mRNA, as well as IL-6, and IL-6R protein. Alcohol consumption along with estrogen treatment further increased steatosis, NFκB translocation, TNF-α mRNA, and IL-6 protein. Conversely, neither estrogen nor ethanol consumption induced IκBα or IκBβ mRNA or protein expression, while testosterone robustly induced these inhibitory proteins regardless of treatment.

Conclusions

Estrogen exposure enhances alcohol-induced liver inflammation, and the anti-inflammatory effects of testosterone in the liver might be related to induction of IκB. Elevated inflammation in response to estrogen may overwhelm the regenerative influence of IL-6 in liver, leading to increased steatosis and greater liver damage.     

地西他滨对K562/A02细胞阿霉素耐药性的影响

 目的: 观察地西他滨(DAC)对人慢性粒细胞白血病耐药细胞株K562/A02阿霉素(ADR)耐药性的影响,探讨其作用的可能机制。方法: 分别或联合应用不同浓度ADR和DAC作用于K562/A02细胞和其亲本细胞株K562,采用CCK-8法检测药物细胞毒性,Sequenom MassARRAY系统结合比色法评价DNA甲基化程度,流式细胞术检测K562/A02细胞细胞周期分布和细胞凋亡率。结果: K562/A02细胞较K562细胞具有显著ADR耐药性,前者ADR作用24 h的IC₅₀约为后者的50倍。而对DAC,在0.5~8 μmol/L作用浓度范围内,K562/A02细胞则较K562细胞更敏感。在相同ADR作用浓度(4.31和17.24 μmol/L)下,联合1 μmol/L DAC处理24 h能显著提高K562/A02细胞对ADR的敏感性,细胞存活率下降(P<0.05)。DAC和ADR均能影响K562/A02细胞的细胞周期进程和细胞凋亡率。1 μmol/L DAC的影响与作用时间相关,在作用24 h时以S期阻滞与细胞早期凋亡率升高为主,48 h时以G₂/M期阻滞与细胞晚期凋亡和坏死率升高为主。ADR则主要表现为浓度依赖性G₂/M期阻滞并诱导细胞晚期凋亡和坏死。两者联用使ADR对细胞周期分布的作用进一步加强,即表现为G₂/M期阻滞更加明显,但对细胞凋亡率的影响并无显著差异。而在基因组甲基化程度上,2种细胞没有显著差异,DAC作用前后也没有显著改变。结论: DAC能增强K562/A02细胞对ADR的敏感性,具有逆转耐药作用,其机制可能与调节K562/A02细胞细胞周期进程、促进细胞凋亡和坏死有关。     

Selection of cytotoxic T lymphocytes against autologous human melanoma from lymph nodes with metastatic melanoma using repeatedin vitro sensitization

Our goal was to determine the cytotoxic activity of effector cells in lymph nodes with metastatic melanoma. Lymphocytes contained within tumor cells from metastatic lymph nodes of two patients were allowed to proliferate in recombinant IL-2 (rIL-2, 100-1,000 units/ml) after 14–21 days of culture. Each set of lymphocytes showed cytotoxicity against autologous melanoma (AM, mean 72%) at effector to target ratio of 201 and K562 cells (mean 60%) using 4-h chromium-51 release assay. Using unlabeled AM and K562, each AM could partially block the activity against K562, but K562 could not block the activity against AM. These activated lymphocytes underwentin vitro sensitization (IVS) with irradiated AM cells and rIL-2 at 2-week intervals. After repeated IVS over about 50 days, each patient's lymphocytes showed cytotoxicity against AM (mean 54%) but not K562 (mean 5%,P < 0.001). These results indicate that different cytotoxic effector cells were present in the early and late phase of lymphocyte tumor culture. Repeated IVS resulted in the selection of specific cytotoxic T lymphocytes. Cold target inhibition assay demonstrated that melanoma cells contained common and individual AM-associated antigen in addition to K562-associated antigens.This work was supported by Biomedical Research Support Grant of the University of Arizona (no. 2S07 RR05675-20), the Elsa U. Pardee Foundation Grant, partly by the Arizona Chronic Disease Research Commission and partly by CA23074 from the National Institutes of Health, Bethesda, 20892, U.S.A.Recipient of the American Cancer Society Clinical Oncology Career Award, 1987–90.     

TAK1 maintains the survival of immunoglobulin λ‐chain‐positive B cells

TAK1 (MAP3K7) mediation of the IκB kinase (IKK) complex?nuclear factor‐κB (NF‐κB) pathway is crucial for the activation of immune response and to perpetuate inflammation. Although progress has been made to understand TAK1 function in the B‐cell receptor (BCR) signaling, the physiological roles of TAK1 in B‐cell development, particularly in the bone marrow (BM), remain elusive. Previous studies suggested that the IKK complex is required for the development of immunoglobulin light chain λ‐positive B cells, but not for receptor editing. In contrast, NF‐κB activity is suggested to be involved in the regulation of receptor editing. Thus, NF‐κB signaling in early B‐cell development is yet to be fully characterized. Therefore, we addressed the role of TAK1 in early B‐cell development. TAK1‐deficient mice showed significant reduction of BM Igλ‐positive B‐cell numbers without any alteration in the BCR editing. Furthermore, the expression of survival factor Bcl‐2 was reduced in TAK1‐deficient BM B cells as assessed by microarray and quantitative PCR analyses. Ex vivo over‐expression of exogenous Bcl‐2 enhanced the survival of TAK1‐deficient Igλ‐positive B cells. TAK1–IKK–NF‐κB signaling contributes to the survival of λ‐chain‐positive B cells through NF‐κB‐dependent anti‐apoptotic Bcl‐2 expression.     

20(R)人参皂甙Rg3逆转K562/ADM细胞MDR及诱导其凋亡的研究

目的　观察抗癌新药 2 0 (R) 人参皂甙Rg3(2 0 (R) ginsenoside ,Rg3)对K5 6 2 /ADM多耐药细胞株逆转MDR机制。方法　应用MTT法确定ADM和Rg3的细胞毒 ;用荧光分光光度仪测定K5 6 2 /ADM细胞内阿霉素 (Adriamycin ,ADM )的浓度 ;流式细胞仪检测细胞凋亡率和表达P 170糖蛋白 (P glycoprotein ,Pgp)的K5 6 2 /ADM的细胞含量。 结果　 (1)K5 6 2 /ADM细胞对阿霉素 (Adriamycin ,ADM )的抗性比K5 6 2细胞高 11倍 ,但两者对Rg3的IC50 分别为 11 76± 0 33μg/ml、10 4 9± 0 30 μg/ml,无显著性差异 (P >0 0 5 )。(2 )无毒剂量和低毒剂量的Rg3能显著提高K5 6 2 /ADM细胞内ADM的浓度 ,使该细胞对ADM的敏感性明显提高。 (3)Rg3对K5 6 2 /ADM细胞有较强的抑制生长和诱导凋亡作用 ,并有时间和浓度依赖性。(4)Rg3对表达P 170糖蛋白的K5 6 2 /ADM细胞的百分含量无显著性影响。结论　 2 0 (R) 人参皂甙Rg3不仅是抑制肿瘤生长和转移的抗瘤剂 ,也是一种有效的MDR逆转剂和细胞凋亡诱导剂     

In vivo and in vitro anti-tumour response of selenium-protein polysaccharide extracted from rich selenium Agaricus blazei

In this study, the anti-tumour activity of selenium-protein polysaccharide (SPP), a water extract of the rich selenium Agaricus blazei, was tested both in vivo and in vitro. The results of in vivo experiments show that SPP at doses of 50 and 100 mg/kg inhibits proliferation of implanted Sarcoma 180 by 22 and 37.69%, respectively, and promotes lymphocyte transformation and natural killer (NK) cells activity in tumour bearing mice. During the in vitro experiment, we treated the tumour and non-tumour bearing mice with SPP, and prepared serum treated with SPP (Serum_SPP). The results show that Serum_SPP, whether from tumour or non-tumour bearing mice, significantly inhibits K562 cells proliferation and induces their apoptosis, and also significantly increases caspase-3 activity of K562 cells. However, the difference in anti-tumour activity of Serum_SPP between tumour and non-tumour bearing mice is significantly different (p<0.01). The results, according to the studies both in vivo and in vitro, imply that SPP extracted from rich selenium A. blazei can inhibit growth of implanted Sarcoma 180 and promote lymphocyte transformation and NK cells activity in vivo. Additionally, Serum_SPP can inhibit proliferation and cause apoptotic morphological changes and the fragmentation of internucleosomal DNA, and increase caspase-3 activity of K562 cells in vitro, which indicates that apoptosis of K562 cells induced by Serum_SPP may be related to up-regulation of caspase-3.     

Activation of human NK cells and monocytes with cisplatin in vitro

Human natural killer (NK) cells and monocytes treated in vitro concomitantly with cisplatin and rIFN-γ enhanced lysis of K562 cells. Lysis was dependent upon the duration of treatment. Cisplatin and rIFN-γ treated monocytes were equally cytotoxic to NK sensitive (K562) and NK resistant (Daudi & Raji) cell lines whereas NK cells were not rendered cytotoxic against NK resistant tumor cells. NK- and monocyte-mediated cytotoxicity against K562 cells was further enhanced when the effector cells were primed with rIFN-γ and were subsequently treated with cisplatin.     

Involvement of IbeA in meningitic Escherichia coli K1-induced polymorphonuclear leukocyte transmigration across brain endothelial cells

Transmigration of neutrophil [polymorphonuclear neutrophil (PMN)] across the blood-brain barrier (BBB) is a critical event in the pathogenesis of bacterial meningitis. We have shown that IbeA is able to induce meningitic Escherichia coli invasion of brain microvascular endothelial cells (BMECs), which constitutes the BBB. In this report, we provide evidence that IbeA and its receptor, vimentin, play a key role in E. coli-induced PMN transmigration across BMEC. In vitro and in vivo studies indicated that the ibeA-deletion mutant ZD1 was significantly less active in stimulating PMN transmigration than the parent strain E44. ZD1 was fully complemented by the ibeA gene and its product. E. coli-induced PMN transmigration was markedly inhibited by withaferin A, a dual inhibitor of vimentin and proteasome. These cellular effects were significantly stimulated and blocked by overexpression of vimentin and its head domain deletion mutant in human BMEC, respectively. Our studies further demonstrated that IbeA-induced PMN migration was blocked by bortezomib, a proteasomal inhibitor and correlated with upregulation of endothelial ICAM-1 and CD44 expression through proteasomal regulation of NFκB activity. Taken together, our data suggested that IbeA and vimentin contribute to E. coli K1-stimulated PMN transendothelial migration that is correlated with upregulation of adhesion molecule expression at the BBB.     

Effect of shark cartilage derived protein on the NK cells activity

Context: Shark cartilage has been used for its beneficial effects on various diseases. There are evidences, that shark cartilage stimulates cellular and humoral immune responses, which makes it an anti-tumor and immunomodulator candidate.

Objective: The immunostimulatory effect of shark cartilage derived proteins on the cytotoxic activity of natural killer (NK) cells from healthy human peripheral blood mononuclear cells was studied.

Material and methods: The shark cartilage was extracted and its bioactive proteins were purified using ion-exchange chromatography (DE-52) and sequential fractionation on Amicon ultrafiltration membranes. The effect of each protein fraction on the modulation of cytotoxic activity of NK cells, as effectors, against K562, as target cells, was assayed by enzymatic lactate dehydrogenase test.

Results: The most immunostimulatory effect on the cytotoxic activity of NK cells was observed for AR10 fraction, containing proteins with molecular weight of about 14.5?kDa on the reducible discontinuous sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Discussion: Among the examined shark cartilage derived proteins, the most immunostimulatory effects on the NK cells cytotoxicity was found for AR10 fraction with molecular weight of about 14?kDa. We propose—the direct interactions of shark cartilage derived proteins with NK cells surface receptors may lead to the enhancing in the cytotoxic activity of NK cells.

Conclusion: Thus AR10 fraction, proteins of about 14.5?kDa, has a novel immunostimulatory effect on the NK cells activity in vitro and if confirmed by in vivo trials, it may lead to its future clinical applications as, immunotherapy of cancer, HIV, and augmentation of host immune system related immunodeficiency disorders.     

Lactobacillus sakei K17, an inducer of IL-10 expression in antigen-presenting cells,attenuates TNBS-induced colitis in mice

To understand the anti-colitic effects of probiotics that up-regulate interleukin (IL)-10 expression in dendritic cells (DCs) and macrophages, we isolated Lactobacillus sakei K17, which potently induced IL-10 expression in DCs and peritoneal macrophages in vitro, among the lactic acid bacteria strains collected from kimchi and investigated its anti-inflammatory effect in mice with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Oral administration of K17 (2?×?10⁹ CFU·mouse^?1·day^?1) in mice with TNBS-induced colitis suppressed colon shortening and myeloperoxidase activity, as well as infiltration of CD86^+?cells into the colon. Treatment with K17 also increased TNBS-suppressed expression of tight junction proteins and IL-10, but inhibited activation of nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinases and expression of tumor necrosis factor α and IL-17. Its effect was comparable with that of sulfasalazine (50?mg/kg), a positive commercial ant-colitic drug. Furthermore, treatment with K17 (1?×?10⁵ CFU/mL) potently inhibited lipopolysaccharide (LPS)-stimulated NF-κB activation in DCs and peritoneal macrophages and restored tight junction protein expression in LPS-stimulated Caco-2 cells. These findings suggest that Lactobacillus sakei K17 may ameliorate colitis by up-regulating the expression of IL-10 and tight junction proteins and inhibiting NF-κB activation.