Licensed human natural killer cells aid dendritic cell maturation via TNFSF14/LIGHT

Interactions between natural killer (NK) cells and dendritic cells (DCs) aid DC maturation and promote T-cell responses. Here, we have analyzed the response of human NK cells to tumor cells, and we identify a pathway by which NK–DC interactions occur. Gene expression profiling of tumor-responsive NK cells identified the very rapid induction of TNF superfamily member 14 [TNFSF14; also known as homologous to lymphotoxins, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes (LIGHT)], a cytokine implicated in the enhancement of antitumor responses. TNFSF14 protein expression was induced by three primary mechanisms of NK cell activation, namely, via the engagement of CD16, by the synergistic activity of multiple target cell-sensing NK-cell activation receptors, and by the cytokines IL-2 and IL-15. For antitumor responses, TNFSF14 was preferentially produced by the licensed NK-cell population, defined by the expression of inhibitory receptors specific for self-MHC class I molecules. In contrast, IL-2 and IL-15 treatment induced TNFSF14 production by both licensed and unlicensed NK cells, reflecting the ability of proinflammatory conditions to override the licensing mechanism. Importantly, both tumor- and cytokine-activated NK cells induced DC maturation in a TNFSF14-dependent manner. The coupling of TNFSF14 production to tumor-sensing NK-cell activation receptors links the tumor immune surveillance function of NK cells to DC maturation and adaptive immunity. Furthermore, regulation by NK cell licensing helps to safeguard against TNFSF14 production in response to healthy tissues.Natural killer (NK) cells play an important role in protecting the host against viral infection and cancer. As well as having potent cytotoxic activity, NK cells are endowed with immunoregulatory activity (1, 2). For example, NK cell activation induces the production of chemokines, such as macrophage inflammatory protein-1α (MIP-1α) and IL-8, and proinflammatory cytokines, such as IFN-γ, GM-CSF, and TNF-α. These molecules regulate the recruitment and activity of numerous immune cell types (1, 2). Importantly, NK cells can promote development of T-cell responses via NK–dendritic cell (DC) interactions that favor both DC maturation and NK-cell activation (3–5), with NK cell-derived IFN-γ skewing T-cell differentiation toward the Th1 phenotype (6, 7).Cytotoxic activity and cytokine production are coupled to signaling pathways downstream of a repertoire of activating and inhibitory receptors; signals from activating receptors (including NKG2D, DNAM-1, and 2B4, as well as the natural cytotoxicity receptors NKp30, NKp44, and NKp46) compete with signals from inhibitory receptors such as the killer cell immunoglobulin-like receptors (KIRs) and CD94/NKG2A heterodimers to regulate activation. In addition, NK cells express CD16, the low-affinity receptor for IgG, conferring antibody-dependent cellular cytotoxicity (8–10). Activation thus coordinates the killing of target cells, the induction of inflammation, and the promotion of adaptive immunity. This potent cytotoxicity and proinflammatory activity must be strictly controlled to minimize damage to healthy tissue. Functional competency of unstimulated NK cells is achieved via a process termed “licensing” or “education” (11–14). Licensing ensures that only those NK cells expressing inhibitory receptors for self-MHC class I can respond to target cells and NK cells that lack inhibitory receptors for self-MHC class I molecules are rendered hyporesponsive, preventing them from attacking healthy cells expressing normal levels of MHC class I molecules.We have analyzed the consequences of human NK cell activation by tumor cells. Our results reveal induction of the TNF superfamily member 14 (TNFSF14), also known as homologous to lymphotoxins, exhibits inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes (LIGHT) (15). We show that activated NK cells produce TNFSF14 in response to different stimuli, that tumor cells induce TNFSF14 production by licensed NK cells, and that TNFSF14-producing NK cells aid DC maturation during NK–DC cross-talk.     

Small molecule probes to quantify the functional fraction of a specific protein in a cell with minimal folding equilibrium shifts

Although much is known about protein folding in buffers, it remains unclear how the cellular protein homeostasis network functions as a system to partition client proteins between folded and functional, soluble and misfolded, and aggregated conformations. Herein, we develop small molecule folding probes that specifically react with the folded and functional fraction of the protein of interest, enabling fluorescence-based quantification of this fraction in cell lysate at a time point of interest. Importantly, these probes minimally perturb a protein’s folding equilibria within cells during and after cell lysis, because sufficient cellular chaperone/chaperonin holdase activity is created by rapid ATP depletion during cell lysis. The folding probe strategy and the faithful quantification of a particular protein’s functional fraction are exemplified with retroaldolase, a de novo designed enzyme, and transthyretin, a nonenzyme protein. Our findings challenge the often invoked assumption that the soluble fraction of a client protein is fully folded in the cell. Moreover, our results reveal that the partitioning of destabilized retroaldolase and transthyretin mutants between the aforementioned conformational states is strongly influenced by cytosolic proteostasis network perturbations. Overall, our results suggest that applying a chemical folding probe strategy to other client proteins offers opportunities to reveal how the proteostasis network functions as a system to regulate the folding and function of individual client proteins in vivo.All proteins are biosynthesized as linear chains, and most need to fold into 3D structures to function. Studies on protein folding in buffers have revealed that a kinetic competition typically exists between protein folding, misfolding, and aggregation. It is the role of the protein homeostasis or proteostasis network in each subcellular compartment to regulate this competition and keep the folded and functional proteome within the physiological concentration range, while minimizing misfolding and aggregation in the face of stresses (1–4). It remains a challenge to discern how the proteostasis network affects the folding of proteins into biologically active conformations required for function in vivo (5).Current methodologies allow for quantification of the partitioning of a protein of interest (POI) between soluble and aggregated states but cannot determine the proportion of the soluble population that is properly folded and functional. Published folding probes have the potential to report on the folded fraction in cells or cell lysate (6–9); however, the extent to which they shift folding equilibria and quantify the folded and functional fraction faithfully has not been studied. Herein, we create POI folding probes by adapting the principle of activity-based protein profiling (10) to quantify the soluble folded and functional fraction of a particular protein in a cell lysate. We seek folding probes that bind to and selectively react with only the folded and functional state of a POI in a cell, leaving the nonfunctional states and other cellular proteins unmodified (Fig. 1A).Open in a separate windowFig. 1.A small molecule folding probe strategy to quantify the soluble folded and functional fraction of a POI in a cell lysate. (A) Overview of the general strategy to selectively covalently label a folded and functional POI without labeling its nonfunctional conformations and other cellular proteins. (B) The experimental scheme to quantify the ratio of the soluble POI that is functional (R_f).Fluorescent folding probes for the de novo-designed enzyme, retroaldolase (RA) (11), and fluorogenic folding probes (12) for the nonenzyme protein, transthyretin (TTR), were developed and scrutinized. We show that destabilized mutant RA and TTR proteins partition into folded and functional as well as misfolded soluble conformations and that this partitioning is sensitive to proteostasis network perturbations. Experiments show that a snapshot of the distribution between folded and functional vs. soluble and misfolded conformational states can be preserved during the small molecule folding probe labeling period, provided that the cellular chaperone holdase activity is sufficient, achieved by rapid ATP depletion in parallel with cell lysis. Sufficient chaperone/chaperonin holdase activity minimizes changes in the folded and functional concentration associated with probe binding and reaction with the POI and renders the relative folding and conjugation rates much less influential.     

Tropical countries may be willing to pay more to protect their forests

Inadequate funding from developed countries has hampered international efforts to conserve biodiversity in tropical forests. We present two complementary research approaches that reveal a significant increase in public demand for conservation within tropical developing countries as those countries reach upper-middle-income (UMI) status. We highlight UMI tropical countries because they contain nearly four-fifths of tropical primary forests, which are rich in biodiversity and stored carbon. The first approach is a set of statistical analyses of various cross-country conservation indicators, which suggests that protective government policies have lagged behind the increase in public demand in these countries. The second approach is a case study from Malaysia, which reveals in a more integrated fashion the linkages from rising household income to increased household willingness to pay for conservation, nongovernmental organization activity, and delayed government action. Our findings suggest that domestic funding in UMI tropical countries can play a larger role in (i) closing the funding gap for tropical forest conservation, and (ii) paying for supplementary conservation actions linked to international payments for reduced greenhouse gas emissions from deforestation and forest degradation in tropical countries.Primary forests—“forests of native species in which there are no clearly visible signs of past or present human activity” (ref. 1, p. 11)—are globally significant repositories of biodiversity (2) and carbon (3). The global area of these forests is declining at an annual percentage rate that is nearly triple the rate for total global forest area (ref. 1, tables 2.4 and 3.3). Virtually all of the loss is occurring in tropical countries (SI Text, section 1). Logging is the main cause of the loss (ref. 1, p. 27), but hunting threatens biodiversity even in primary forests with intact tree cover (4, 5).Protecting primary tropical forests is a core mission of several international institutions created since the early 1990s, including the Convention on Biological Diversity (CBD), the Global Environment Facility (GEF), and the UN Collaborative Program on Reducing Emissions from Deforestation and Forest Degradation in Developing Countries (REDD). However, the CBD failed to achieve its goal of significantly reducing biodiversity loss by 2010 (6); international funding for biodiversity protection through the GEF and other mechanisms is below commitments made at the 1992 Earth Summit (7) and the amounts required to achieve the CBD’s 2020 protection targets (8); and REDD has not advanced beyond a readiness phase (www.un-redd.org). Relying on international mechanisms to fund protection of primary tropical forests does not look like a winning strategy.Here, we argue that economic development during the past 20–25 y has raised public demand for forest protection within tropical countries, but the level of protection supplied by tropical country governments has not kept pace. We focus on the dynamics of conservation and development within relatively wealthier developing countries: The group that is classified by the World Bank as upper-middle income (UMI). As we will show, the majority of primary forest area in tropical countries is found in these countries. We hypothesize that public willingness to pay (WTP) to protect forests has reached a relatively high level in UMI countries, leading to greater support for local conservation nongovernmental organizations (NGOs) and prompting governments to boost forest protection efforts—but not as much as the public would like. This gap between domestic demand and domestic supply of forest protection has two important implications: Domestic funding might be sufficient to cover the costs of additional protection in some, and perhaps many, tropical countries; and international funding might be able to leverage more domestic funding than it currently does.Although many cross-country studies in the environmental Kuznets curve (EKC) literature have investigated the effect of rising national income on deforestation (9), none has considered the effect on primary forests. This gap matters because deforestation, unlike primary forest loss, results mainly from agricultural conversion, not logging (1, 10). A few cross-country studies have considered the effect of national income on creation of protected areas (11–16), but with mixed findings on the significance of the effect. A second and larger group of studies has used surveys to measure WTP for biodiversity conservation by domestic populations within particular countries. Most of these studies have failed to detect a significant income effect (P < 0.05) (17, 18). Metaanalyses of these studies estimate income effects that are generally positive (protection increases with income) but not necessarily statistically significant (17, 18).We extended this prior work by coupling two research approaches: a broad-brush statistical analysis of the association between a standard measure of economic development, i.e., per capita gross national income (GNI), and a large set of cross-country conservation indicators (CIs); and a focused investigation of forest protection in a particular UMI country, Malaysia. The statistical analysis spanned 12 indicators from 10 diverse sources (Materials and Methods and SI Text, section 1). The indicators pertained to public environmental preferences, conservation NGOs, and government action (conservation spending, protected area establishment). These indicators relate more directly to our hypothesis about domestic demand and domestic supply of forest protection than do the deforestation rates analyzed by EKC studies. We limited the samples to countries classified as tropical by the UN Food and Agriculture Organization Forestry Department (ref. 19, data table 2) and paid special attention to differences between tropical countries in the UMI group and ones in lower income groups.The Malaysian case study allowed us to examine more closely the linkages from rising household income to increased household WTP, NGO engagement, and government protective action, and thereby uncover reasons for the underprovision of forest protection relative to household preferences. The case concerned Belum–Temengor, a 300,000-ha forested region in the state of Perak (Fig. 1). This region contains the largest area of primary forest in Peninsular Malaysia outside a national park. Our research included a population-representative survey of 1,261 rural and urban households in the Malaysian state of Selangor and the federal territory of Kuala Lumpur during 2010 (Materials and Methods). We used choice experiments (20, 21) to estimate household WTP to protect Belum–Temengor against logging and poaching (SI Text, section 2). Information from the case study enabled us to compare the public’s aggregate WTP for protection to current protection expenditures and to discuss why there is a gap between the two.Open in a separate windowFig. 1.Locations of Belum–Temengor (site of forest protection plans in choice experiments) and Selangor and Kuala Lumpur (site of household survey; the black dot is Kuala Lumpur) within Peninsular Malaysia (light gray). Lines show Malaysian state boundaries. Sources: base map, GADM database (www.gadm.org); Belum–Temengor boundaries, Forest Research Institute Malaysia.     

Does glyceryl trinitrate cause central sympatholytic effects? Insights from a case of baroreflex failure

Whether part of the blood pressure lowering effects of glyceryl trinitrate (GTN) is the result of centrally mediated reduction in sympathetic activity is debated. In humans, baroreflex activity potentially obscures the central sympatholytic effects of GTN. We examined this in a routine clinical tilt test in a patient with baroreflex failure secondary to previous neck radiotherapy. With reduced baroreflex function we observed an exaggerated fall in blood pressure and reduced sympathetic activity with GTN, supporting a peripheral vasodilation and central sympatholytic effect.     

NPM1, FLT3-ITD,CEBPA, and c-kit mutations in 312 Chinese patients with de novo acute myeloid leukemia

Objectives

To explore NPM1, FLT3-ITD, CEBPA, and c-kit mutations in patients with acute myeloid leukemia (AML) from Chinese population.

Methods

In this study, we retrospectively analyzed the prevalence and clinical pro?le of NPM1, FLT3-ITD, CEBPA, and c-kit mutations in 312 patients with de novo AML.

Results

The frequencies of NPM1, FLT3-ITD, c-kit, and CEBPA mutations were 15.4, 14.0, 7.64, and 25.6%, respectively. The occurrence rate of NPM1 mutations increased with age in patients younger than 60 years. NPM1, c-kit, and CEBPA mutations were all associated with French-American-British subtypes. Patients with NPM1 mutations and FLT3-ITD presented with higher peripheral white blood cell counts and marrow blast percentages.

Conclusion

Both this and previous studies may suggest low frequencies of NPM1 and FLT3-ITD mutations in AML patients from the Chinese population, and they may have a synergistic function in stimulating proliferation of leukemia cells.     

Percutaneous edge‐to‐edge repair for common atrioventricular valve regurgitation in a patient with heterotaxy syndrome,single ventricle physiology,and unbalanced atrioventricular septal defect

Congenital heart disease patients, specifically with unbalanced atrioventricular septal defects and common atrioventricular valves requiring single ventricle palliation, have substantial morbidity and mortality. Atrioventricular valve regurgitation (AVVR) is associated with poor outcomes in single ventricle patients, and many of them require surgical treatment of AVVR in their lifetimes. We describe a unique case of transcatheter edge‐to‐edge valve repair using the MitraClip system (Abbott, Chicago, IL) in a single ventricle patient with severe common AVVR.     

奥美定隆乳注射患者术后取出的手术方式分析

探讨奥美定隆乳注射患者术后取出的手术方式。对我科收治的奥美定注射患者,根据其临床表现、B超和MRI检查分成包膜硬结型、散在团块型、液态游走型及混合型,对不同类型的患者采取不同的手术方式。比较其愈合时间、术后并发症及复发情况。乳腔镜手术在伤口愈合时间上显著短于乳晕切口包块取出术。液态游走型或混合型患者行乳腔镜下包块取出术,术后伤口感染、疾病复发率显著高于乳腺切除组。包膜硬结型、散在团块型可行乳腔镜包块取出术、乳晕切口包块取出术或乳腺部分切除术,以尽量保留乳腺的完整,液态游走型或混合型应行乳腺全切术以彻底根治。