Natural Killer (NK) cells recognize and destroy tumors and virus-infected cells in an antibody-independent manner. The regulation of NK cells is mediated by activating and inhibiting receptors on the ...NK cell surface. One important family of activating receptors is the natural cytotoxicity receptors (NCRs) which include NKp30, NKp44 and NKp46. The NCRs initiate tumor targeting by recognition of heparan sulfate on cancer cells. This study aims to elucidate heparan sulfate structural motifs that are important for NCR binding. Microarray and surface plasmon resonance experiments with a small library of heparan sulfate/heparin oligosaccharides helped to clarify the binding preferences of the three NCRs. We demonstrate that the NCRs interact with highly charged HS/heparin structures, but differ in preferred modification patterns and chain lengths. The affinity of NKp30 and NKp44 for synthetic HS/heparin is approximately one order of magnitude higher than the affinity of NKp46. We further show the relevance of synthetic HS/heparin for the binding of NCRs to tumor cells and for NCR-mediated activation of natural killer cells. In conclusion, NCRs recognize different microdomains on heparan sulfate with different affinities.
Attachment of a conjugate assembled from a novel fluorinated carbonic anhydrase inhibitor and rhodamine B onto dehydroabietylamine (DHA) or cyclododecylamine led to first-in-class conjugates of good ...cytotoxicity; thereby IC50 values (from SRB assays; employed tumor cell lines A2780, A2780Cis, A549, HT29, MCF7, and non-malignant human fibroblasts CCD18Co) between 0.2 and 0.7 μM were found. Both conjugates showed similar cytotoxic activity but the dehydroabietylamine derived conjugate outperformed its cyclododecyl analog in terms of tumor cell/non-tumor cell selectivity. Both conjugates accumulate intracellular, and the DHA conjugate was able to overcome drug resistance which is effective independent of the expression status of carbonic anhydrase IX.
Display omitted
•Dehydroabietylamine was converted into a sulfonamide/rhodamine B conjugate.•This conjugate was tested for cytotoxic activity with a panel of human cancer cells.•This conjugate as well as a cyclododecyl analog held IC50 < 1 μM.•Both conjugates were able to overcome drug resistance and accumulated intracellular.
Natural killer (NK) cells play a role in the early control and natural course of hepatitis C virus (HCV) infection. NK cell function is regulated by a multitude of receptors, including activating ...NKp46 receptor. However, reports on NKp46 in hepatitis C are controversial. Therefore, we investigated the hepatic recruitment and function of NKp46(+) NK cells, considering differential surface expression of NKp46 resulting in NKp46High and NKp46Dim subsets. Intra‐ and extrahepatic NK‐cell subsets from HCV‐infected patients were characterized by flow cytometry. Cytotoxic activity and interferon‐gamma (IFN‐γ) secretion were studied using K‐562, P815, and primary hepatic stellate cells as targets. Anti‐HCV activity of NK‐cell subsets was studied using the replicon system. Density of NKp46 surface expression clearly segregated NKp46Dim and NKp46High subsets, which differed significantly with respect to the coexpression of maturation markers and NK‐cell receptors. More important, NKp46High NK cells showed a higher cytolytic activity and stronger IFN‐γ secretion than NKp46Dim NK cells. Accordingly, NKp46High NK cells efficiently blocked HCV replication in vitro. Blocking experiments confirmed an important role for the NKp46 receptor. Furthermore, we found an intrahepatic accumulation of NKp46High NK cells. Of note, high cytolytic activity of NKp46High NK cells was also confirmed in the intrahepatic NK‐cell population, and the frequency of intrahepatic NKp46High NK cells was inversely correlated with HCV‐RNA levels and fibrosis stage. Conclusions: NKp46High expression defines a specific NK‐cell subset that may be involved in both the suppression of HCV replication and HCV‐associated liver damage underpinning the role of NK cells in the immunopathogenesis of HCV. (HEPATOLOGY 2012)
The activating natural cytotoxicity receptor NKp30 is critical for natural killer (NK) cell function and tumor immune surveillance. The natural cytotoxicity receptor‐3 (NCR3) gene is transcribed into ...several splice variants whose physiological relevance is still incompletely understood. In this study, we investigated the role of NKp30 and its major ligand B7 homolog 6 (B7‐H6) in patients with hepatocellular carcinoma (HCC). Peripheral blood NK cell phenotype was skewed toward a defective/exhausted immune profile with decreased frequencies of cells expressing NKp30 and natural killer group 2, member D and an increased proportion of cells expressing T‐cell immunoglobulin and mucin‐domain containing‐3. Moreover, NKp30‐positive NK cells had a reduced expression of NCR3 immunostimulatory splice variants and an increased expression of the inhibitory variant in patients with advanced tumor, resulting in deficient NKp30‐mediated functionality. Tumor‐infiltrating lymphocytes showed a prevalent inhibitory NKp30 isoform profile, consistent with decreased NKp30‐mediated function. Of note, there were significant differences in the cytokine milieu between the neoplastic and the surrounding non‐neoplastic tissue, which may have further influenced NKp30 function. Exposure of NK cells to B7‐H6‐expressing HCC cells significantly down‐modulated NKp30, that was prevented by small interfering RNA–mediated knockdown, suggesting a role for this ligand in inhibiting NKp30‐mediated responses. Interestingly, B7‐H6 expression was reduced in HCC tissue and simultaneously augmented as a soluble form in HCC patients, particularly those with advanced staging or larger nodule size. Conclusion: These findings provide evidence in support of a role of NKp30 and its major ligand in HCC development and evolution.
Wikstroemia indica (L.) C. A. Mey. (Thymelaceae) is well documented for use in traditional medicine, but scientific studies on this plant species are lacking. Hence, its phytochemical and ...pharmacological properties were investigated using different methods. The total phenolic (66.00 ± 0.44 mg GAE/g), flavonoid (69.96 ± 0.32 mg RE/g), phenolic acid (32.59 ± 1.29 mg CE/g) and flavonol (4.46 ± 0.07 mg CAE/g) content of the methanolic extract (ME) was highest when compared to the ethyl acetate extract (EAE) and aqueous extract (AE) respectively. Altogether, 31 compounds were identified by HPLC (High Performance Liquid Chromatography) method. The DPPH (2,2-diphenylpicrylhydrazyl), ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), FRAP (Ferric reducing antioxidant power assay), assays demonstrated that the ME had the highest antioxidant potential with recorded values of 188.38 ± 0.22, 421.31 ± 14.54, 397.10 ± 2.68 and 651.19 ± 16.90 mg TE/g respectively. Regarding the acetylcholinesterase, the ME and the EAE were the best inhibitors (3.13 ± 0.11 and 3.11 ± 0.14 mg GALAE/g). Only the EAE was active against the butyrylcholinesterase enzyme with recorded value of 7.05 ± 0.14 mg GALAE/g. The tyrosinase inhibitory assays demonstrated that the extract inhibitory potential was in the following order: ME (117.63 ± 2.53 mg KAE/g) > EAE (112.57 ± 0.85 mg KAE/g) > AE (13.65 ± 0.87 mg KAE/g). The alpha amylase inhibitory potential of the EA and ME were the same (0.68 ± 0.01 mmol ACAE/g) and better than the AE (0.10 ± 0.01 mmol ACAE/g). HT 29 treated with W. indica in aqueous started to exhibit cell cytotoxicity from 500 µg/mL. The IC50 value exhibited at 786.14 µg/mL. W. indica can be developed into extracts to promote general health pending toxicological studies that show the plant is entirely safe for human consumption.
Display omitted
•Chemical profile and biological effects of Wikstroemia indica extracts were investigated.•31 compounds were identified in the tested extracts.•The methanol extract had great antioxidant properties.•The extracts exhibited great cytotoxic properties.•The plants might be utilized as a natural source of biologically-active compounds.
Natural killer (NK) cells are a part of the innate immune system that functions mainly to kill transformed and infected cells. Their activity is controlled by signals derived from a panel of ...activating and inhibitory receptors. The natural cytotoxicity receptors (NCRs): NKp30, NKp44, and NKp46 (NCR1 in mice) are prominent among the activating NK cell receptors and they are, notably, the only NK-activating receptors that are able to recognize pathogen-derived ligands. In addition, the NCRs also recognize cellular ligands, the identity of which remains largely unknown. In this review, we summarize the current knowledge regarding viruses that are recognized by the NCRs, focusing on the diverse immune-evasion mechanisms employed by viruses to escape this detection. We also discuss the unique role the NCRs have in regulating NK cell activity with particular emphasis on the in vivo function of NKp46/NCR1.
The N-glycans attached to the Fab and Fc domains play distinct roles in modulating the functions of antibodies. However, posttranslational site-selective modifications of glycans in antibodies and ...other multiply glycosylated proteins remain a challenging task. Here, we report a chemoenzymatic method that permits independent manipulation of the Fab and Fc N-glycans, using cetuximab as a model therapeutic monoclonal antibody. Taking advantage of the substrate specificity of three endoglycosidases (Endo-S, Endo-S2, and Endo-F3) and their glycosynthase mutants, together with an unexpected substrate site-selectivity of a bacterial α1,6-fucosidase from Lactobacillus casei (AlfC), we were able to synthesize an optimal homogeneous glycoform of cetuximab in which the heterogeneous and immunogenic Fab N-glycans were replaced with a single sialylated N-glycan, and the core-fucosylated Fc N-glycans were remodeled with a nonfucosylated and fully galactosylated N-glycan. The glycoengineered cetuximab demonstrated increased affinity for the FcγIIIa receptor and significantly enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity.
PDF
