Distinct Innate Immunity Pathways to Activation and Tolerance in Subgroups of Chronic Lymphocytic Leukemia with Distinct Immunoglobulin Receptors

Subgroups of patients with chronic lymphocytic leukemia (CLL) have distinct expression profiles of Toll-like receptor (TLR) pathway–associated genes. To test the hypothesis that signaling through innate immunity receptors may influence the behavior of the malignant clone, we investigated the functional response triggered by the stimulation of TLRs and NOD2 in 67 CLL cases assigned to different subgroups on the basis of immunoglobulin heavy variable (IGHV ) gene usage, IGHV gene mutational status or B-cell receptor (BcR) stereotypy. Differences in the induction of costimulatory molecules and/or apoptosis were observed in mutated versus unmutated CLL. Different responses were also identified in subsets with stereotyped BcRs, underscoring the idea that “subset-biased” innate immunity responses may occur independently of mutational status. Additionally, differential modulation of kinase activities was induced by TLR stimulation of different CLL subgroups, revealing a TLR7-tolerant state for cases belonging to stereotyped subset #4. The distinct patterns of TLR/NOD2 functional activity in cells from CLL subgroups defined by the molecular features of the clonotypic BcRs might prove relevant for elucidating the immune mechanisms underlying CLL natural history and for defining subgroups of patients who might benefit from treatment with specific TLR ligands.     

Five Conditions Commonly Used to Down-regulate Tor Complex 1 Generate Different Physiological Situations Exhibiting Distinct Requirements and Outcomes

Five different physiological conditions have been used interchangeably to establish the sequence of molecular events needed to achieve nitrogen-responsive down-regulation of TorC1 and its subsequent regulation of downstream reporters: nitrogen starvation, methionine sulfoximine (Msx) addition, nitrogen limitation, rapamycin addition, and leucine starvation. Therefore, we tested a specific underlying assumption upon which the interpretation of data generated by these five experimental perturbations is premised. It is that they generate physiologically equivalent outcomes with respect to TorC1, i.e. its down-regulation as reflected by TorC1 reporter responses. We tested this assumption by performing head-to-head comparisons of the requirements for each condition to achieve a common outcome for a downstream proxy of TorC1 inactivation, nuclear Gln3 localization. We demonstrate that the five conditions for down-regulating TorC1 do not elicit physiologically equivalent outcomes. Four of the methods exhibit hierarchical Sit4 and PP2A phosphatase requirements to elicit nuclear Gln3-Myc¹³ localization. Rapamycin treatment required Sit4 and PP2A. Nitrogen limitation and short-term nitrogen starvation required only Sit4. G₁ arrest-correlated, long-term nitrogen starvation and Msx treatment required neither PP2A nor Sit4. Starving cells of leucine or treating them with leucyl-tRNA synthetase inhibitors did not elicit nuclear Gln3-Myc¹³ localization. These data indicate that the five commonly used nitrogen-related conditions of down-regulating TorC1 are not physiologically equivalent and minimally involve partially differing regulatory mechanisms. Further, identical requirements for Msx treatment and long-term nitrogen starvation raise the possibility that their effects are achieved through a common regulatory pathway with glutamine, a glutamate or glutamine metabolite level as the sensed metabolic signal.     

Compartmentalization of Distinct cAMP Signaling Pathways in Mammalian Sperm

Fertilization competence is acquired in the female tract in a process known as capacitation. Capacitation is needed for the activation of motility (e.g. hyperactivation) and to prepare the sperm for an exocytotic process known as acrosome reaction. Although the HCO₃⁻-dependent soluble adenylyl cyclase Adcy10 plays a role in motility, less is known about the source of cAMP in the sperm head. Transmembrane adenylyl cyclases (tmACs) are another possible source of cAMP. These enzymes are regulated by stimulatory heterotrimeric G_s proteins; however, the presence of G_s or tmACs in mammalian sperm has been controversial. In this study, we used Western blotting and cholera toxin-dependent ADP-ribosylation to show the G_s presence in the sperm head. Also, we showed that forskolin, a tmAC-specific activator, induces cAMP accumulation in sperm from both WT and Adcy10-null mice. This increase is blocked by the tmAC inhibitor SQ22536 but not by the Adcy10 inhibitor KH7. Although G_s immunoreactivity and tmAC activity are detected in the sperm head, PKA is only found in the tail, where Adcy10 was previously shown to reside. Consistent with an acrosomal localization, G_s reactivity is lost in acrosome-reacted sperm, and forskolin is able to increase intracellular Ca²⁺ and induce the acrosome reaction. Altogether, these data suggest that cAMP pathways are compartmentalized in sperm, with G_s and tmAC in the head and Adcy10 and PKA in the flagellum.     

Removal of Linear and Branched Alkylphenols from Aqueous Solutions with Horseradish Peroxidase

Alkylphenols were effectively treated with horseradish peroxidase at pH 7.0 and 30 °C in the presence of H₂O₂ and poly(ethylene glycol) irrespective of the relative position or isomeric form of the alkyl chains. Water-insoluble oligomer precipitates were readily filtered out after enzymatic treatment, and transparent and colorless solutions were obtained for all p- and m-alkylphenols used.     

Insect Antiviral Innate Immunity: Pathways,Effectors, and Connections

Insects are infected by a wide array of viruses some of which are insect restricted and pathogenic, and some of which are transmitted by biting insects to vertebrates. The medical and economic importance of these viruses heightens the need to understand the interaction between the infecting pathogen and the insect immune system in order to develop transmission interventions. The interaction of the virus with the insect host innate immune system plays a critical role in the outcome of infection. The major mechanism of antiviral defense is the small, interfering RNA pathway that responds through the detection of virus-derived double-stranded RNA to suppress virus replication. However, other innate antimicrobial pathways such as Imd, Toll, and Jak-STAT and the autophagy pathway have also been shown to play important roles in antiviral immunity. In this review, we provide an overview of the current understanding of the main insect antiviral pathways and examine recent findings that further our understanding of the roles of these pathways in facilitating a systemic and specific response to infecting viruses.     

SIRT3 is Pro-Apoptotic and Participates in Distinct Basal Apoptotic Pathways

SIRT3, one of seven mammalian sirtuins, is a NAD-dependent deacetylase. SIRT3 localises to mitochondria where it deacetylates and thus activates acetyl-CoA synthetase 2 (AceCS2), indicating a role for SIRT3 in metabolism. Here we provide evidence that SIRT3 also impacts upon apoptosis and cell growth control. Using RNAi under basal (non-stress) conditions we show that SIRT3 is required for apoptosis induced by selective silencing of Bcl-2 in HCT116 human epithelial cancer cells. Identical treatment of ARPE19 epithelial non-cancer cells induces G1 growth arrest which also proved to be SIRT3-dependent. Previously we have identified SIRT1 and JNK2 as constitutive suppressors of apoptosis in HCT116 cells. We now demonstrate that SIRT3 functions in JNK2-regulated apoptosis but is dispensable for SIRT1-regulated apoptosis. SIRT3 is also dispensable for stress-induced apoptosis. Thus the pro-apoptotic functioning of SIRT3 is selectively coupled with defined pathways regulating cell survival under basal conditions.     

三种细菌DNA及CpG寡核苷酸抗肿瘤免疫的比较实验研究

用大肠埃希菌、铜绿假单胞菌和双歧杆菌的DNA与CpG寡聚核苷酸 (CpGoligodeoxynucleotides ,CpGODN)对荷瘤鼠进行治疗 ,比较 3种细菌DNA及CpGODN的抗肿瘤免疫作用 ,以筛选最好的细菌DNA及CpG序列用于肿瘤治疗。分别提取 3种细菌DNA并合成 2条CpGODN序列 ,在肝癌HcaFA3细胞株荷瘤小鼠模型上进行免疫治疗 ,以存活期、抑瘤率、NK/Mφ细胞活性及细胞因子 (IFN γ、TNF α、IL 2、IL 12等 )分泌活性等为指标 ,比较 3种细菌DNA之间及CpGODN的抗肿瘤免疫效果。 3种细菌DNA均能使荷瘤鼠的存活期延长 1倍以上 ,但三者之间无显著性差异 (P >0 .0 5 )。抑瘤率为 6 4 .0 3%～ 79.5 0 % (P <0 .0 1) ,而CpGODN的抑瘤率为 4 5 %～ 5 1%之间 (P <0 .0 5 ) ,3种细菌DNA以及CpGODN均能显著增强NK细胞和Mφ细胞的杀伤活性 (P <0 .0 5 ) ,但细菌DNA在诱导细胞因子 (IL 2、IFN、TNF等 )以及下调肿瘤细胞端粒酶活性方面 ,CpGODN作用强于细菌DNA。     

多种HIVB′/C亚型基因在复制型DNA疫苗中表达及免疫效果研究

为了解多种HIVB′/C亚型基因在复制型DNA疫苗中表达水平及对免疫效果影响,使用复制型DNA疫苗载体pSCK2,分别构建了7种含单种或多种HIVB′/C亚型基因gagpol、gp160和rtn(rev、tat和nef融合基因)的DNA疫苗质粒。免疫荧光检测表明,Gag、Gp160、Rev、Tat和Nef蛋白均能从相应7种DNA疫苗中表达,单基因表达质粒中的基因表达水平和双基因表达质粒中IRES上游的基因表达水平普遍较好。小鼠免疫后,Gag能诱发较高的抗体滴度,Gp160、Pol和RTN的抗体滴度很低;比较研究显示,Gag单独表达和Gag与RTN双表达的质粒均能诱发较好的Gag抗体反应,但Gag与Gp160双表达质粒免疫或含Gag、Gp160和RTN质粒联合免疫的Gag抗体反应均较弱。ELISPOT检测表明,7种DNA疫苗单独或不同组合方式免疫均能诱发针对Gag、Pol、Gp160、Tat和Nef的细胞免疫反应;单基因表达质粒单独免疫诱发的细胞免疫反应最好,含gagpol和gp160双基因表达质粒单独免疫或与含其它基因质粒联合免疫诱发的Gag、Pol和Gp160细胞免疫明显低于含Gagpol或Gp160单基因质粒诱发的相应免疫反应,但诱发的Tat和Nef细胞免疫与相应单基因质粒免疫组无明显差别。本研究结果显示,不同表达构建体的多种HIVB'/C亚型的基因gagpol、gp160和rtn均能从pSCK2质粒中较好地表达;不同基因结构和不同组合免疫的优化,特别是含gag和gp160基因疫苗联合免疫程序的进一步优化对提高其免疫效果是必要的。     

Brassinosteroids Regulate Plant Growth through Distinct Signaling Pathways in Selaginella and Arabidopsis

Brassinosteroids (BRs) are growth-promoting steroid hormones that regulate diverse physiological processes in plants. Most BR biosynthetic enzymes belong to the cytochrome P450 (CYP) family. The gene encoding the ultimate step of BR biosynthesis in Arabidopsis likely evolved by gene duplication followed by functional specialization in a dicotyledonous plant-specific manner. To gain insight into the evolution of BRs, we performed a genomic reconstitution of Arabidopsis BR biosynthetic genes in an ancestral vascular plant, the lycophyte Selaginella moellendorffii. Selaginella contains four members of the CYP90 family that cluster together in the CYP85 clan. Similar to known BR biosynthetic genes, the Selaginella CYP90s exhibit eight or ten exons and Selaginella produces a putative BR biosynthetic intermediate. Therefore, we hypothesized that Selaginella CYP90 genes encode BR biosynthetic enzymes. In contrast to typical CYPs in Arabidopsis, Selaginella CYP90E2 and CYP90F1 do not possess amino-terminal signal peptides, suggesting that they do not localize to the endoplasmic reticulum. In addition, one of the three putative CYP reductases (CPRs) that is required for CYP enzyme function co-localized with CYP90E2 and CYP90F1. Treatments with a BR biosynthetic inhibitor, propiconazole, and epi-brassinolide resulted in greatly retarded and increased growth, respectively. This suggests that BRs promote growth in Selaginella, as they do in Arabidopsis. However, BR signaling occurs through different pathways than in Arabidopsis. A sequence homologous to the Arabidopsis BR receptor BRI1 was absent in Selaginella, but downstream components, including BIN2, BSU1, and BZR1, were present. Thus, the mechanism that initiates BR signaling in Selaginella seems to differ from that in Arabidopsis. Our findings suggest that the basic physiological roles of BRs as growth-promoting hormones are conserved in both lycophytes and Arabidopsis; however, different BR molecules and BRI1-based membrane receptor complexes evolved in these plants.     

Distinct Unfolding and Refolding Pathways of Lipid Transfer Proteins LTP1 and LTP2

Plant non-specific lipid transfer proteins (ns-LTPs) comprise two families, LTP1s and LTP2s, all structurally stabilized by four native disulfide bonds. Solution and crystal structures of both LTP1s and LTP2s from various plants have been determined. Despite the similarities of their biological function and backbone folds, the biophysical properties of LTP1s and LTP2s differ significantly. In this report, the mechanisms of unfolding and refolding of rice LTP1 and LTP2 have been investigated using the technique of disulfide bonds scrambling. LTP1 is shown to unfold and refold via predominant species of partially structured intermediates. Four isomers of partly unfolded and extensively unfolded LTP1 were identified, isolated and their disulfide structures were determined. By contrast, unfolding and refolding of LTP2 adopt a (close to) two-state mechanism, and undergo a reversible conversion between the native and a single extensively unfolded isomer without accumulation of any significant intermediate.     

Distinct Regulation of Cytoplasmic Calcium Signals and Cell Death Pathways by Different Plasma Membrane Calcium ATPase Isoforms in MDA-MB-231 Breast Cancer Cells

Plasma membrane calcium ATPases (PMCAs) actively extrude Ca(2+) from the cell and are essential components in maintaining intracellular Ca(2+) homeostasis. There are four PMCA isoforms (PMCA1-4), and alternative splicing of the PMCA genes creates a suite of calcium efflux pumps. The role of these different PMCA isoforms in the control of calcium-regulated cell death pathways and the significance of the expression of multiple isoforms of PMCA in the same cell type are not well understood. In these studies, we assessed the impact of PMCA1 and PMCA4 silencing on cytoplasmic free Ca(2+) signals and cell viability in MDA-MB-231 breast cancer cells. The PMCA1 isoform was the predominant regulator of global Ca(2+) signals in MDA-MB-231 cells. PMCA4 played only a minor role in the regulation of bulk cytosolic Ca(2+), which was more evident at higher Ca(2+) loads. Although PMCA1 or PMCA4 knockdown alone had no effect on MDA-MB-231 cell viability, silencing of these isoforms had distinct consequences on caspase-independent (ionomycin) and -dependent (ABT-263) cell death. PMCA1 knockdown augmented necrosis mediated by the Ca(2+) ionophore ionomycin, whereas apoptosis mediated by the Bcl-2 inhibitor ABT-263 was enhanced by PMCA4 silencing. PMCA4 silencing was also associated with an inhibition of NFκB nuclear translocation, and an NFκB inhibitor phenocopied the effects of PMCA4 silencing in promoting ABT-263-induced cell death. This study demonstrates distinct roles for PMCA1 and PMCA4 in the regulation of calcium signaling and cell death pathways despite the widespread distribution of these two isoforms. The targeting of some PMCA isoforms may enhance the effectiveness of therapies that act through the promotion of cell death pathways in cancer cells.     

Prior Population Immunity Reduces the Expected Impact of CTL-Inducing Vaccines for Pandemic Influenza Control

Vaccines that trigger an influenza-specific cytotoxic T cell (CTL) response may aid pandemic control by limiting the transmission of novel influenza A viruses (IAV). We consider interventions with hypothetical CTL-inducing vaccines in a range of epidemiologically plausible pandemic scenarios. We estimate the achievable reduction in the attack rate, and, by adopting a model linking epidemic progression to the emergence of IAV variants, the opportunity for antigenic drift. We demonstrate that CTL-inducing vaccines have limited utility for modifying population-level outcomes if influenza-specific T cells found widely in adults already suppress transmission and prove difficult to enhance. Administration of CTL-inducing vaccines that are efficacious in "influenza-experienced" and "influenza-naive" hosts can likely slow transmission sufficiently to mitigate a moderate IAV pandemic. However if neutralising cross-reactive antibody to an emerging IAV are common in influenza-experienced hosts, as for the swine-variant H3N2v, boosting CTL immunity may be ineffective at reducing population spread, indicating that CTL-inducing vaccines are best used against novel subtypes such as H7N9. Unless vaccines cannot readily suppress transmission from infected hosts with naive T cell pools, targeting influenza-naive hosts is preferable. Such strategies are of enhanced benefit if naive hosts are typically intensively mixing children and when a subset of experienced hosts have pre-existing neutralising cross-reactive antibody. We show that CTL-inducing vaccination campaigns may have greater power to suppress antigenic drift than previously suggested, and targeting adults may be the optimal strategy to achieve this when the vaccination campaign does not have the power to curtail the attack rate. Our results highlight the need to design interventions based on pre-existing cellular immunity and knowledge of the host determinants of vaccine efficacy, and provide a framework for assessing the performance requirements of high-impact CTL-inducing vaccines.     

不同低氧运动模式对大鼠免疫功能的影响

目的:探讨不同低氧运动模式对大鼠免疫功能的影响。方法:30只SD大鼠随机分为常氧运动组(对照组)、高住高练组和高住低练组,对照组大鼠每天在常氧环境下生活23 h,训练1 h;高住高练组每天在低氧环境生活23 h,训练1 h;高住低练组每天在低氧环境生活12 h,常氧环境下生活11 h,训练1 h。全血分析仪分析全血指标,流式细胞仪检测CD4~ 和CD8~ 占有的百分比。结果:与高住低练组及对照组比较,高住高练组大鼠血液红细胞计数(RBC)和血红蛋白水平(Hb)升高,CD4~ T细胞百分数降低,CD8~ T细胞百分数升高,CD4~ /CD8~ 比值下降,差异均具有统计学意义(P<0.05)。对照组大鼠、高住高练组和高住低练组ELISPOT斑点数依次为(230.0±10.6)/2×10~5个脾细胞、(56.5±6.8)/2×10~5个脾细胞和(89.3±9.8)/2×10~5个脾细胞,差异有统计学意义(P<0.05)。结论:低氧运动可以提高机体氧运输能力,但是可能会损害机体免疫功能。     

Distinct Modes of Inhibition by Sclerostin on Bone Morphogenetic Protein and Wnt Signaling Pathways

Sclerostin is expressed by osteocytes and has catabolic effects on bone. It has been shown to antagonize bone morphogenetic protein (BMP) and/or Wnt activity, although at present the underlying mechanisms are unclear. Consistent with previous findings, Sclerostin opposed direct Wnt3a-induced but not direct BMP7-induced responses when both ligand and antagonist were provided exogenously to cells. However, we found that when both proteins are expressed in the same cell, sclerostin can antagonize BMP signaling directly by inhibiting BMP7 secretion. Sclerostin interacts with both the BMP7 mature domain and pro-domain, leading to intracellular retention and proteasomal degradation of BMP7. Analysis of sclerostin knock-out mice revealed an inhibitory action of sclerostin on Wnt signaling in both osteoblasts and osteocytes in cortical and cancellous bones. BMP7 signaling was predominantly inhibited by sclerostin in osteocytes of the calcaneus and the cortical bone of the tibia. Our results suggest that sclerostin exerts its potent bone catabolic effects by antagonizing Wnt signaling in a paracrine and autocrine manner and antagonizing BMP signaling selectively in the osteocytes that synthesize simultaneously both sclerostin and BMP7 proteins.     

Distinct Annular Oligomers Captured along the Assembly and Disassembly Pathways of Transthyretin Amyloid Protofibrils

Background

Defects in protein folding may lead to severe degenerative diseases characterized by the appearance of amyloid fibril deposits. Cytotoxicity in amyloidoses has been linked to poration of the cell membrane that may involve interactions with amyloid intermediates of annular shape. Although annular oligomers have been detected in many amyloidogenic systems, their universality, function and molecular mechanisms of appearance are debated.

Methodology/Principal Findings

We investigated with high-resolution in situ atomic force microscopy the assembly and disassembly of transthyretin (TTR) amyloid protofibrils formed of the native protein by pH shift. Annular oligomers were the first morphologically distinct intermediates observed in the TTR aggregation pathway. Morphological analysis suggests that they can assemble into a double-stack of octameric rings with a 16±2 nm diameter, and displaying the tendency to form linear structures. According to light scattering data coupled to AFM imaging, annular oligomers appeared to undergo a collapse type of structural transition into spheroid oligomers containing 8–16 monomers. Disassembly of TTR amyloid protofibrils also resulted in the rapid appearance of annular oligomers but with a morphology quite distinct from that observed in the assembly pathway.

Conclusions/Significance

Our observations indicate that annular oligomers are key dynamic intermediates not only in the assembly but also in the disassembly of TTR protofibrils. The balance between annular and more compact forms of aggregation could be relevant for cytotoxicity in amyloidogenic disorders.     

Regulation of Endothelial Cell Proliferation and Vascular Assembly through Distinct mTORC2 Signaling Pathways

Mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates a diverse array of cellular processes, including cell growth, survival, metabolism, and cytoskeleton dynamics. mTOR functions in two distinct complexes, mTORC1 and mTORC2, whose activities and substrate specificities are regulated by complex specific cofactors, including Raptor and Rictor, respectively. Little is known regarding the relative contribution of mTORC1 versus mTORC2 in vascular endothelial cells. Using mouse models of Raptor or Rictor gene targeting, we discovered that Rictor ablation inhibited vascular endothelial growth factor (VEGF)-induced endothelial cell proliferation and assembly in vitro and angiogenesis in vivo, whereas the loss of Raptor had only a modest effect on endothelial cells (ECs). Mechanistically, the loss of Rictor reduced the phosphorylation of AKT, protein kinase Cα (PKCα), and NDRG1 without affecting the mTORC1 pathway. In contrast, the loss of Raptor increased the phosphorylation of AKT despite inhibiting the phosphorylation of S6K1, a direct target of mTORC1. Reconstitution of Rictor-null cells with myristoylated AKT (Myr-AKT) rescued vascular assembly in Rictor-deficient endothelial cells, whereas PKCα rescued proliferation defects. Furthermore, tumor neovascularization in vivo was significantly decreased upon EC-specific Rictor deletion in mice. These data indicate that mTORC2 is a critical signaling node required for VEGF-mediated angiogenesis through the regulation of AKT and PKCα in vascular endothelial cells.