Treatment for children with high-risk neuroblastoma with anti-disialoganglioside mAb ch14.18, IL-2, and GM-CSF plus 13-cis-retinoic acid after myeloablative chemotherapy improves survival, but 40 % of patients still relapse during or after this therapy. The microenvironment of high-risk neuroblastoma tumors includes macrophages, IL-6, and TGFβ1. We hypothesized that this microenvironment suppresses anti-tumor functions of natural killer (NK) cells and that lenalidomide, an immune-modulating drug, could overcome suppression.
Methods
Purified NK cells were cultured with IL-2, neuroblastoma/monocyte-conditioned culture medium (CM), IL-6, TGFβ1, and lenalidomide in various combinations and then characterized using cytotoxicity (direct and antibody-dependent cell-mediated cytotoxicity), cytokine, flow cytometry, and Western blotting assays. Anti-tumor activity of NK cells with lenalidomide, ch14.18, or both was evaluated with a xenograft model of neuroblastoma.
Results
CM from neuroblastoma/monocyte co-cultures contains IL-6 and TGFβ1 that suppress IL-2 activation of NK cell cytotoxicity and IFNγ secretion. IL-6 and TGFβ1 activate the STAT3 and SMAD2/3 pathways in NK cells and suppress IL-2 induction of cytotoxicity, granzymes A and B release, perforin expression, and IFNγ secretion. Lenalidomide blocks IL-6 and TGFβ1 activation of these signaling pathways and inhibits their suppression of NK cells. Neuroblastoma cells in NOD/SCID mice exhibit activated STAT3 and SMAD2/3 pathways. Their growth is most effectively inhibited by co-injected peripheral blood mononuclear cells (PBMC) containing NK cells when mice are treated with both ch14.18 and lenalidomide.
Conclusion
Immunotherapy with anti-tumor cell antibodies may be improved by lenalidomide, which enhances activation of NK cells and inhibits their suppression by IL-6 and TGFβ1. 相似文献
While numerous small ubiquitin‐like modifier (SUMO) conjugated substrates have been identified, very little is known about the cellular signalling mechanisms that differentially regulate substrate sumoylation. Here, we show that acetylation of SUMO E2 conjugase Ubc9 selectively downregulates the sumoylation of substrates with negatively charged amino acid‐dependent sumoylation motif (NDSM) consisting of clustered acidic residues located downstream from the core ψ‐K‐X‐E/D consensus motif, such as CBP and Elk‐1, but not substrates with core ψ‐K‐X‐E/D motif alone or SUMO‐interacting motif. Ubc9 is acetylated at residue K65 and K65 acetylation attenuates Ubc9 binding to NDSM substrates, causing a reduction in NDSM substrate sumoylation. Furthermore, Ubc9 K65 acetylation can be downregulated by hypoxia via SIRT1, and is correlated with hypoxia‐elicited modulation of sumoylation and target gene expression of CBP and Elk‐1 and cell survival. Our data suggest that Ubc9 acetylation/deacetylation serves as a dynamic switch for NDSM substrate sumoylation and we report a previously undescribed SIRT1/Ubc9 regulatory axis in the modulation of protein sumoylation and the hypoxia response. 相似文献
A neuronal F‐box protein FSN‐1 regulates Caenorhabditis elegans neuromuscular junction development by negatively regulating DLK‐mediated MAPK signalling. In the present study, we show that attenuation of insulin/IGF signalling also contributes to FSN‐1‐dependent synaptic development and function. The aberrant synapse morphology and synaptic transmission in fsn‐1 mutants are partially and specifically rescued by reducing insulin/IGF‐signalling activity in postsynaptic muscles, as well as by reducing the activity of EGL‐3, a prohormone convertase that processes agonistic insulin/IGF ligands INS‐4 and INS‐6, in neurons. FSN‐1 interacts with, and potentiates the ubiquitination of EGL‐3 in vitro, and reduces the EGL‐3 level in vivo. We propose that FSN‐1 may negatively regulate insulin/IGF signalling, in part, through EGL‐3‐dependent insulin‐like ligand processing. 相似文献
Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important swine pathogens because it is highly infectious and causes economic losses due to decreased pig productivity. In this study, the 603 bp complete major envelope protein encoding gene (ORF5) of 32 field PRRSV isolates from Vietnam collected during 2008–2012 were sequenced and analyzed. Multiple nucleotide (nt) and deduced amino acid (aa) alignments of ORF5 were performed on the 32 isolates: the representative strains (European and North American genotypes), Chinese strains available in GenBank and vaccine strains licensed for use in Vietnam. The results showed 94.8–100.0% nt identity and 94.0–100% aa similarity among the 32 isolates. These isolates shared similarities with the prototype of the North American PRRSV strain (VR‐2332; nt 87.8–89.3%, aa 87.5–90.0%), and Lelystat virus, the prototype of the European PRRSV strain (LV; nt 61.1–61.9%, aa 55.1‐57.0%). There was greater similarity with QN07 (nt 96.5‐98.5%, aa 96.0‐99.0%) from the 2007 PRRS outbreak in QuangNam Province, CH‐1a (nt 93.2–95.1%, 91.5–93.5%) isolated in China in 1995 and JXA1 (nt 96.5–98.6%, aa 95.0–98.0%), the highly pathogenic strain from China isolated in 2006. The Vietnamese isolates were more similar to JXA1‐R (nt 96.5–98.6%, aa 95.0–98.0%), the strain used in Chinese vaccines, than to Ingelvac MLV/BSL‐PS (nt 87.2–89.0%, aa 86.0–89.0%). Phylogenetic analysis showed that the 32 isolates were of the North American genotype and classified into sub‐lineage 8.7. This sub‐lineage contains highly pathogenic Chinese PRRSV strains. This study documents genetic variation in circulating PRRSV strains and could assist more effective use of PRRS vaccines in Vietnam. 相似文献
AbstractCognitive repair by insulin-like growth factor-I (IGF-I) through activation of insulin-like growth factor-I receptor (IGF-IR) is well established, but not used for clinical therapy due to its link to cancer. We hypothesize that IGF-IR activation rather than IGF-I per se may be essential for cognitive repair and attempted to identify ligands from traditional Chinese medicine (TCM) with drug-like potential towards IGF-IR. TCM ligands, 3-(2-carboxyphenyl)-4(3H)-quinazolinone from Isatisin digotica, (+)-N-methyllaurotetanine from Lindera aggregate, and (+)-1(R)-Coclaurine from Nelumbonucifera Gaertn, exhibited high binding affinities and good blood brain barrier (BBB) penetration crucial for accessing IGF-IR. Stable complex formation of the candidates was observed during molecular dynamics (MD) simulation. Interactions with Leu975 and Gly1055 or Asp1056 were important for ligand binding. Amino acid distance analysis revealed residues 974/975, 984–986, 996–1006, 1040–1056, and 1122–1135 as “hotspots” for ligand binding in IGF-IR. Versatile entry pathways for the TCM candidates suggest high accessibility to the binding site. Blockage of the binding site opening by the TCM candidates limits binding site access by other compounds. Multiple linear regression (R2?=?0.9715), support vector machine (R2?=?0.9084), Bayesian network (R2?=?0.8233) comparative molecular field analysis (CoMFA, R2?=?0.9941), and comparative molecular similarity indices analysis (CoMSIA, R2?=?0.9877) models consistently suggest that the TCM candidates might exert bioactivity on IGF-IR. Contour of representative MD conformations to CoMFA and CoMSIA maps exhibits similar results. Properties including BBB passage, evidence of ability to form stable complexes with IGF-IR by MD simulation, and predicted bioactivity suggest that the TCM candidates have drug-like properties and might have potential as cognitive-enhancing drugs.An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:38相似文献
Similarly to Helicobacter pylori but unlike Vibrio cholerae O1/O139, Campylobacter jejuni is non‐motile at 20°C but highly motile at ≥37°C. The bacterium C. jejuni has one of the highest swimming speeds reported (>100 μm/s), especially at 42°C. Straight and spiral bacterial shapes share the same motility. C. jejuni has a unique structure in the flagellate polar region, which is characterized by a cup‐like structure (beneath the inner membrane), a funnel shape (opening onto the polar surface) and less dense space (cytoplasm). Other Campylobacter species (coli, fetus, and lari) have similar motility and flagellate polar structures, albeit with slight differences. This is especially true for Campylobacter fetus, which has a flagellum only at one pole and a cup‐like structure composed of two membranes. 相似文献
Using a microchannel assay, we demonstrate that cells adopt distinct signaling strategies to modulate cell migration in different physical microenvironments. We studied α4β1 integrin–mediated signaling, which regulates cell migration pertinent to embryonic development, leukocyte trafficking, and melanoma invasion. We show that α4β1 integrin promotes cell migration through both unconfined and confined spaces. However, unlike unconfined (2D) migration, which depends on enhanced Rac1 activity achieved by preventing α4/paxillin binding, confined migration requires myosin II–driven contractility, which is increased when Rac1 is inhibited by α4/paxillin binding. This Rac1–myosin II cross talk mechanism also controls migration of fibroblast-like cells lacking α4β1 integrin, in which Rac1 and myosin II modulate unconfined and confined migration, respectively. We further demonstrate the distinct roles of myosin II isoforms, MIIA and MIIB, which are primarily required for confined and unconfined migration, respectively. This work provides a paradigm for the plasticity of cells migrating through different physical microenvironments. 相似文献
Energy failure and oxidative stress have been implicated in the pathogenesis of ischemia. Here, we report a potential link between cytosolic phospholipase A2 (cPLA2) activation and energy failure/oxidative stress‐induced astrocyte damage involving reactive oxygen species (ROS), protein kinase C‐α (PKC‐α), Src, Raf, and extracellular signal‐regulated kinase (ERK) signaling and concurrent elevation of endogenous chelatable zinc. Energy failure and oxidative stress were produced by treating astrocytes with glycolytic inhibitor iodoacetate and glutathione chelator diethylmaleate, respectively. Diethylmaleate and iodoacetate in combination caused augmented damage to astrocytes in a time‐ and concentration‐dependent manner. The cell death caused by diethylmaleate/iodoacetate was accompanied by increased ROS generation, PKC‐α membrane translocation, Src, Raf, ERK, and cPLA2 phosphorylation. Pharmacological studies revealed that these activations all contributed to diethylmaleate/iodoacetate‐induced astrocyte death. Intriguingly, the mobilization of endogenous chelatable zinc was observed in diethylmaleate/iodoacetate‐treated astrocytes. Zinc appears to act as a downstream mediator in response to diethylmaleate/iodoacetate treatment because of the attenuating effects of its chelator N,N,N′,N′‐tetrakis(2‐pyridylmethyl)ethylenediamine. These observations indicate that ROS/PKC‐α, Src/Raf/ERK signaling and cPLA2 are active participants in diethylmaleate/iodoacetate‐induced astrocyte death and contribute to a vicious cycle between the depletion of ATP/glutathione and the mobilization of chelatable zinc as critical upstream effectors in initiating cytotoxic cascades.