Posttranslational processing of defensins in immature human myeloid cells.

Human neutrophil promyelocytes synthesize, process, and package several microbicidal proteins, including the highly abundant defensins, into their azurophil granules. As deduced from their cDNA sequences, defensins are initially synthesized as 94 amino acid (aa) precursors that must undergo extensive processing. We performed metabolic labeling studies of defensin synthesis in the human promyelocytic cell line HL-60 and in chronic myeloid leukemia cells, and showed that preprodefensins are processed to mature 29 to 30 aa defensins over 4 to 24 hours via two major intermediates: a 75 aa prodefensin generated by the cleavage of the signal sequence, and a 56 aa prodefensin that results from a subsequent preaspartate proteolytic cleavage. Almost all of the 75 aa form was found in the cytoplasmic/microsomal fraction, whereas the 56 aa prodefensin and mature defensins predominated in the granule-enriched fraction. The 75 aa prodefensin was also selectively released into the culture supernatant. Treatment of HL-60 cells with monensin, chloroquine, or ammonium chloride, substances that neutralize acidic subcellular compartments, partially blocked conversion of the 75 aa prodefensin into 56 aa prodefensin, but did not increase the extracellular release of the 75 aa form. Further studies will be required to determine the role of this processing pathway in subcellular targeting to azurophil granules and avoidance of autocytotoxicity.     

Multispecific lymphoid cell surface receptors.

When mice are sequentially immunized with two antigens to give an oligoclonal "double-binding" antibody response, there is a concomitant increase of "double-binding" cell surface receptors on their splenic lymphocytes. Competition studies suggest that the capacity to bind the two ligands, bovine pancreatic ribonuclease (EC 3.1.4.22) and a 2,4-dinitrophenyl (DNP) derivative, is a function of the same molecules. In ribo-nuclease-primed mice, an early response to bovine gamma globulin containing an average of 60 Dnp groups per molecule is the appearance of an increasing number of cells bearing surface receptors binding both ribonuclease and Dnp. Later, these double-binding cells are diluted by cells that bind Dnp, but not ribonuclease. The analogous phenomenon is observed when the two antigens are used in reverse order. While other reports suggest that there may be several different receptors in relatively undifferentiated cells from unimmunized mice, it seems likely that cells committed to antibody production carry a predominant multispecific cell surface immunoglobulin receptor.     

防御素的抗肿瘤研究

当代肿瘤的治疗仍有许多问题未能解决防御素引入肿瘤墓因治疗已成为一个有前景的新途径.防御素(defensins)是存在于哺乳动物多形核中性粒细胞、巨噬细胞、小肠Peneth细胞中的一组同源性很高的生物多肤类,由29一34     

Multispecific targeting of glioblastoma with tumor microenvironment-responsive multifunctional engineered NK cells

Tumor antigen heterogeneity, a severely immunosuppressive tumor microenvironment (TME) and lymphopenia resulting in inadequate immune intratumoral trafficking, have rendered glioblastoma (GBM) highly resistant to therapy. To address these obstacles, here we describe a unique, sophisticated combinatorial platform for GBM: a cooperative multifunctional immunotherapy based on genetically engineered human natural killer (NK) cells bearing multiple antitumor functions including local tumor responsiveness that addresses key drivers of GBM resistance to therapy: antigen escape, immunometabolic reprogramming of immune responses, and poor immune cell homing. We engineered dual-specific chimeric antigen receptor (CAR) NK cells to bear a third functional moiety that is activated in the GBM TME and addresses immunometabolic suppression of NK cell function: a tumor-specific, locally released antibody fragment which can inhibit the activity of CD73 independently of CAR signaling and decrease the local concentration of adenosine. The multifunctional human NK cells targeted patient-derived GBM xenografts, demonstrated local tumor site–specific activity in the tissue, and potently suppressed adenosine production. We also unveil a complex reorganization of the immunological profile of GBM induced by inhibiting autophagy. Pharmacologic impairment of the autophagic process not only sensitized GBM to antigenic targeting by NK cells but promoted a chemotactic profile favorable to NK infiltration. Taken together, our study demonstrates a promising NK cell–based combinatorial strategy that can target multiple clinically recognized mechanisms of GBM progression simultaneously.

Glioblastoma (GBM) is the most common and deadliest malignant type of primary brain tumor (1). GBM patients are poorly responsive to traditional treatments, resulting in a grim prognosis that has only modestly improved over the past several decades, motivating the hunt for new treatment approaches (2). So far, chimeric antigen receptor (CAR)-engineered natural killer (NK) cells targeting single GBM antigens—EGFR, EGFRvIII, or ErbB2/HER2—have been limited to the use of NK cell lines, and the overall response rates have been disappointingly low and inconsistent (3–5). These responses appear to mirror the clinical hurdles of single antigen-targeted CAR-T therapies for GBM (6–9). CAR-T cells, administered to target single GBM antigens via intracavitary, intraventricular, or intravenous routes, have so far resulted in inconclusive durable responses (8).Preclinical and patient data have pointed to the heterogeneity of the GBM tumor microenvironment (TME) as a uniquely complex obstacle to overcome ( 10). This is reflected in immunotherapies tested so far having struggled to improve GBM patient overall survival (OS) in phase III clinical trials (11, 12). GBM induces localized lymphopenia to drive disease progression and resist treatment (13). In addition, the tumor’s heterogeneity is broad, with each of the known GBM subtypes—classical, mesenchymal, neural, and proneural—displaying diverse genetic and epigenetic signatures associated with distinct and variable cell plasticities (14). Not surprisingly, the outgrowth of antigen escape variants has been recorded clinically with most GBM-associated antigens to date, resulting in immune evasion and resistance to treatment (7, 15). And though strategies including dual antigen-targeting or programmable tumor-sensing CARs—so far primarily in the context of adoptive T cell therapy—have been evaluated preclinically to combat such evasion, GBM employs mechanisms beyond antigen escape to avoid targeting. Treatment evasion by GBM is fueled by a heavily immunosuppressive, hypoxic TME, which provides a niche unfavorable to NK cell effector function (16). A subset of GBM cells, glioma stem-like cells (GSCs), contribute to treatment resistance and are poorly recapitulated by conventional GBM model cell lines, including U87MG (17). Metabolic and functional pathways, moreover, converge to fuel the tumor’s invasiveness by driving exhaustion of immune cells (18). For instance, immunometabolic dysregulation of NK cell function in GBM is driven in part by the activity of ecto-5′-nucleotidase (CD73). CD73 is a hypoxic ectoenzyme that we and others have found to be associated with a negative prognosis and has emerged as an attractive clinical target (19, 20). In addition, we have previously shown that the CD73-driven accumulation of extracellular adenosine (ADO) leads to significant purinergic signaling–mediated impairment of NK cell activity (21, 22).Although they are among the most abundant lymphocytes found within the GBM TME, NK cells are still present in insufficient amounts in these tumors and exhibit a highly dysfunctional phenotype (23, 24). The need to recapitulate NK cell function lost to multiple complex mechanisms not only presents a significant challenge to traditional CAR-NK therapy but requires a greater presence specifically within the tumor tissue to mount meaningful clinical responses.Here, we describe an example of a multifunctional, engineered human NK cell–based therapy for glioblastoma developed around the programmed targeting of three clinically recognized pathways of GBM progression: antigen escape, immunometabolic suppression, and poor intratumoral NK cell presence. We achieved dual antigen recognition by modifying NK cells with multi-CARs to target disialoganglioside (GD2) and ligands to NK group 2D (NKG2D), which are widely expressed on human GBM (25, 26). Within the same NK cells, we engineered the concomitant local release of an antibody fragment that impairs immunosuppressive purinergic signaling by blocking the activity of CD73 via the cleavage of a tumor-specific linker. This cleavage is dependent on the activity of proteases that are up-regulated in the tumor microenvironment (27). Such local release is able to avoid systemic toxicities owing to its tumor-specific activation that occurs independently of CAR-based signaling.We report the homing of such multifunctional NK cells was enhanced when administered in conjunction with autophagy inhibitors in patient-derived GBM xenografts. Disabling autophagy further revealed a sophisticated and complex reorganization of anti-GBM immunological responses which could contribute to enhanced CAR-NK effector function. The clinical efficacy of adding an autophagy inhibitor to GBM therapy has shown that such treatments are clinically safe and well tolerated (28). We reveal a nuanced and potentially important role for autophagy inhibitors in adoptive human NK therapy.These studies aim to expand the repertoire of GBM-targeting NK cell–based immunotherapy and describe a first example of addressing, simultaneously, the challenge of tumor antigen heterogeneity, an immunosuppressive TME, and insufficient intratumoral trafficking of NK cells.     

The role of defensins in virus-induced asthma

Respiratory viruses appear to play a central role in asthma pathogenesis. In addition to a major role in triggering asthma exacerbations, viral infections early in life may play a role in disease development. Although defensins were initially identi.ed as antimicrobial peptides, recent studies have demonstrated that they have a much broader range of actions that are of relevance to virally induced asthma. Defensins are not only induced during viral infections, they exert direct antiviral actions against some enveloped viruses, and also can impair viral infection of cells. In addition, defensins modulate the activity of several cell types that contribute to innate immunity, including mast cells, epithelial cells, natural killer cells, and dendritic cells. They also have the capacity to modulate adaptive immunity and to contribute to airway remodeling. In this article, recent advances in these areas are reviewed in the context of the potential role of defensins in virus-induced asthma.     

Mechanisms of disease: defensins in gastrointestinal diseases

Defensins are endogenous antibiotics with microbicidal activity against Gram-negative and Gram-positive bacteria, fungi, viruses and protozoa. In the gastrointestinal tract, defensins help regulate the composition and number of colonizing microbes, and protect the host from food-borne and water-borne pathogens. In health, the normal host relationship with the commensal luminal microbiota is beneficial, but the same commensal bacteria could have a pathogenic role in inflammatory diseases. A disturbance in antimicrobial defense, as provided by Paneth cells of the small intestine, seems to be a critical factor in the pathogenesis of ileal Crohn's disease, an inflammatory disease of the intestinal tract. The disruption of the critical balance between antimicrobial peptides and luminal bacteria might also explain other gastrointestinal infections and diseases. Elucidating the underlying mechanisms involved in the regulation and biology of defensins could open up new therapeutic avenues.     

The role of defensins in lung biology and therapy

Innate host defence, involving both cellular and humoral mediators, is a prominent function of the human airways. Cellular mediators of innate immunity include dendritic cells, natural killer cells, cytotoxic T cells, macrophages and neutrophils, while humoral mediators of innate immunity consist of components of the epithelial lining fluid (ELF) covering the airways. Microbicidal substances in the ELF can selectively disrupt bacterial cell walls and membranes, sequester microbial nutrients or act as decoys for microbial attachment. Antimicrobial components of airway secretions include lysozymes, lactoferrin, secretory leukoprotease inhibitor, defensins and cathelicidins. Defensins are the most widely studied family of antimicrobial peptides present in airway fluid. Humans produce at least 10 different defensin molecules, six alpha-defensins and four beta-defensins similar in structure and function. Direct evidence that defensins have central roles in host defense has only recently become available. Some defensins and defensin-like molecules could serve as templates for the development of pulmonary pharmaceuticals. As potential therapeutics, they possess several desirable properties, including the ability to kill a broad spectrum of micro-organisms while permitting little development of microbial resistance. Many peptides can also neutralize effects of lipopolysaccharide on macrophages and other host defense cells and decrease the release of proinflammatory cytokines thereby giving protection against septic shock. Protegrin-1 is a minidefensin isolated from pig leukocytes and has proved to be an attractive template for large-scale development of antibacterials. One such protegrin analog, iseganan is in phase III clinical trials for the treatment of oral mucositis secondary to systemic chemotherapy. Other prospective uses of iseganan include control of respiratory pathogens in patients with cystic fibrosis and reduction of oral bacteria to prevent ventilator-associated pneumonia. However, in order to advance the production and clinical testing of peptide-based therapeutics, technical hurdles of synthesizing large quantities of complexly folded peptides must be first overcome. Strategies to develop potent peptide-based microbicides are promising in the struggle against increasingly resistant pathogens.     

Detecting Multispecific Patterns in the Catch Composition of a Fisheries-Independent Longline Survey

Understanding the main factors that regulate species composition in fisheries is of utmost importance for developing efficient management strategies, particularly under the scope of ecosystem-based conservation approaches. This study used multivariate statistics to analyze catch data collected during a ~10-year, fishery-independent, standardized longline survey conducted in coastal waters (<20-m isobaths) off Recife, northeastern Brazil. A redundancy analysis (RDA) was performed to assess the influence of spatiotemporal, environmental, and bioecological variables on the variability in longline catch composition and to identify similarly distributed groups of species. Additionally, an analysis of similarities (ANOSIM) was conducted to investigate the likeness among the multispecific groups and identify the most influential variables. A total of 1,295 specimens representing 29 species of teleosts, elasmobranchs, and sea turtles were caught, but most species (62.0%) were little represented (<1%) in the catch composition. The RDA model indicated that the catch composition was significantly influenced by habitat type, behavior, trophic level, year, site, water transparency, month, and sea surface temperature; bioecological variables provided the greatest contribution to explain the variability in catch composition. The ANOSIM revealed that marine catfishes, moray eels, and Nurse Shark Ginglymostoma cirratum were the most similar in their relation to several spatiotemporal and environmental variables. The patterns reported herein might be useful to improve coastal fisheries management because they present the species that are influenced by similar drivers and the main factors underlying their respective catch rates. Therefore, this approach could be a potentially useful tool for lessening the number of biological dimensions, which frequently limit the capacity to implement effective management strategies in multispecies fisheries.

Received December 21, 2016; accepted June 20, 2017     

防御素与肺部疾病的研究进展

防御素是先天性免疫和获得性免疫系统的重要组成部分,已成为生物医学研究的热点。本文主要总结防御素与肺部疾病相关的内容,对防御素在呼吸系统的表达与肺部感染、肺损伤、肺结核、肺纤维化、COPD、支气管哮喘的关系进行简述。     

Flow cytometric analysis of defensins in blood and marrow neutrophils

Polymorphonuclear neutrophils (PMN) are vital in host defense against microbial infections. This study provides a flow cytometric method for the quantitative analysis of microbicidal peptides (defensins) in cells of PMN lineage. Rabbit neutrophil peptides, NP-2 and NP-5, were measured in all PMN and in subpopulations of PMN expressing 1-selectin. PMN lineage counts were made on Wright's-stained blood smears and marrow cytospins. Immunoreactivity for NP-2, and NP-5 was detected by using the alkaline phosphatase anti-alkaline phosphatase technique. The results show that marrow PMN express higher levels of NP-2 and NP-5 than blood PMN, p < 0.001 and that these levels are associated with elevated numbers of myeloid precursors. In both blood and marrow, NP-2 occurs in two PMN subpopulations and the mean fluorescence intensity of NP-2 is consistently higher than that of NP-5. Increased levels of defensins are observed in circulating PMN depicting the most 1-selectin p < 0.05. Immunocytochemical results indicate that PMN defensins reside in cytoplasmic granules and are not constitutively expressed on the cell surface. Furthermore, defensins are not detected in monocytes, eosinophils, lymphocytes and erythrocytes. The flow cytometric method described here provides a novel means of quantitating host natural defenses, allows the characterization of PMN subpopulations and has clinical applications.     

The role of defensins in the pathogenesis of chronic-inflammatory bowel disease

Defensins are endogenous antimicrobial peptides with a broad activity spectrum. Even at micromolar concentrations gramnegative and grampositive bacteria, but also mycobacteria, as well as fungi (candida), viruses (herpes) and protozoa (giardia lamblia) are destroyed. As part of the innate immune system defensins are expressed by the intestinal epithelium and contribute to the maintenance of the mucosal barrier. This barrier appears to be defective in inflammatory bowel diseases since on one hand, the immune response is directed against the "normal" luminal bacterial flora and on the other hand, mucosal adherent and invasive bacteria have been observed in these diseases. A defective defensin expression may well explain these phenomena. Indeed, Crohn's disease of the terminal ileum, especially if associated with a NOD2 mutation, is characterised by a diminished alpha-defensin (human defensin 5 and 6) expression, and in inflamed Crohn's colitis, in contrast to ulcerative colitis, the beta-defensin (human beta-defensins 2 and 3) response is reduced. Through a deficient chemical mucosal barrier this defect could lead to increased bacterial invasion into the intestinal mucosa and might well explain an adequate inflammatory response. Although the final proof that this deficient defensin response leads to a reduced antibacterial activity of the intestinal mucosa is still lacking, the most plausible concept of pathogenesis of Crohn's disease is a defensin deficiency syndrome.     

Roles of antimicrobial peptides such as defensins in innate and adaptive immunity

A number of antimicrobial peptides such as defensins have multiple functions in host defence. Defensins are produced not only by phagocytic cells and lymphocytes, but also by the epithelial cell lining of the gastrointestinal and genitourinary tracts, the tracheobronchial tree, and keratinocytes. Some are produced constitutively, whereas others are induced by proinflammatory cytokines and exogenous microbial products. Defensins produced by cells in the course of innate host defence serve as signals which initiate, mobilise, and amplify adaptive immune host defences. Administration of defensins with antigens to mice enhances both cellular (Th1-dependent) and humoral (Th2-dependent) cytokine production and immune responses. Linkage of defensins to weak tumour antigens potentiates their immunoadjuvant effects. Defensins use multiple cellular receptors, which endows them with the capacity to marshall adaptive host defences against microbial invaders.     

Defensins and inflammation: The role of defensins in inflammatory bowel disease

Defensins are antimicrobial peptides produced at a variety of epithelial surfaces. In the intestinal tract, they contribute to host immunity and assist in maintaining the balance between protection from pathogens and tolerance to normal flora. However, attenuated expression of defensins compromises host immunity and hence may alter the balance toward inflammation. Altered defensin production is suggested to be an integral element in the pathogenesis of inflammatory bowel disease (IBD). Evidence for this is shown in Crohn's disease where reduced α-defensin levels are seen in patients with ileal disease and reduced β-defensin levels in those with colonic involvement. Further evidence is provided by research linking nucleotide oligomerization domain 2 (NOD2) mutations and deficient defensin expression. However, alternate studies suggest that NOD2 status and defensin expression are independent, and that defensin deficiency is due to mucosal surface destruction as a result of inflammatory changes, indicating that reduced defensin expression is a symptom of the disease and not the cause. Although it is clear that defensin expression is altered in IBD, it is less clear whether defensin deficiency is implicated in the pathogenesis of IBD or is a consequence of the disease process. The aim of this article is to review the current knowledge of defensins in IBD and discuss their potential role in IBD pathogenesis.     

Paneth cell defensins and the regulation of the microbiome

Recently, our laboratory demonstrated that Paneth cell defensins, innate antimicrobial peptides that contribute to mucosal host defense, are able to regulate the composition of the intestinal bacterial microbiome. Using complementary mouse models of defensin deficiency (MMP7-/-) and surplus (HD5+/+), we noted defensin-dependent reciprocal shifts in the dominant bacterial species of the small intestine, without changes in total bacterial numbers. In addition, mice that expressed HD5 showed a significant loss of segemented filamentous bacteria (SFB), resulting in reduced numbers of Th17 cells in the lamina propria. This data showed a novel role for PC defensins in intestinal homeostasis, by regulation of the small intestinal microbiome. The microbiome plays an essential role in mediating host physiology, metabolism, and immune response. The ability of PC defensins to regulate the composition of the biome suggests a much broader importance of these innate immune effectors than previously considered. In this addendum, the role of PC defensins in the regulation of the intestinal microbiome is reviewed, and discussed in the context of recent evidence that highlights the important role of PCs and defensins in the pathophysiology of inflammatory bowel disease.     

防御素在溃疡性结肠炎结肠组织中的表达及意义

目的研究防御素（HNP13）、白细胞介素-8（IL-8）在溃疡性结肠炎（UC）结肠组织中的表达分布以及与UC病变范围和病变程度的关系，探讨其在UC发病机制中的作用；明确IL-8与HNP1-3的相关性。方法用免疫组化法检测36例活动期UC及30例正常对照石蜡包埋组织中HNP1-3及IL-8的表达情况。结果UC组HNP1-3及IL-8均为阳性表达。对照组均为阴性或弱阳性表达。两者在UC组的表达与对照组相比，差异均有统计学意义（P〈0．01）。UC病变范围越广、程度越重，HNP13及IL-8表达的阳性率越高。UC患者受累黏膜的HNP1-3和IL-8水平明显高于未受累黏膜。且HNP1-3和IL-8表达具有明显的相关性（rs=0．957，P〈0．01）。结论HNP1-3的表达与IL-8呈正相关且均参与了UC的发生和发展；HNP1-3可能在UC局部结肠组织破坏和病理变化中起了更重要的作用；HNP1-3、IL-8反映了疾病的炎症程度，并且可作为病情严重程度评定的指标。     

NOD2 and defensins: translating innate to adaptive immunity in Crohn's disease

The nucleotide-binding oligomerisation protein 2 (NOD2) is a sensor for bacterial muramyl dipeptide, which ensures ileal expression of antimicrobial peptides (so-called alpha-defensins) and promotes cytokine and chemokine production by immunocytes and enterocytes. Defective NOD2 signaling pathway and impaired expression of defensins were inextricably linked to the pathogenesis of Crohn's disease, a common form of inflammatory bowel disease. NOD2 and defensin deficiency at the level of the epithelial barrier and gut-associated lymphoid tissue may favour Crohn's disease by failing to protect from enteropathogens and to instruct adaptive immune response in the gut micro-environment. Herein, we provide an overview on the key role of NOD2 and defensins in antigen-presenting function of dendritic cells and antigen-specific immunity. We also outline the urgent need for a better understanding of the regulators of NOD2 function and defensin biogenesis to support the development of a rational immunostimulatory treatment for restoring long-lasting immunity in Crohn's disease.     

Gefitinib induces myeloid differentiation of acute myeloid leukemia

Cure rates for patients with acute myeloid leukemia (AML) remain low despite ever-increasing dose intensity of cytotoxic therapy. In an effort to identify novel approaches to AML therapy, we recently reported a new method of chemical screening based on the modulation of a gene expression signature of interest. We applied this approach to the discovery of AML-differentiation-promoting compounds. Among the compounds inducing neutrophilic differentiation was DAPH1 (4,5-dianilinophthalimide), previously reported to inhibit epidermal growth factor receptor (EGFR) kinase activity. Here we report that the Food and Drug Administration (FDA)-approved EGFR inhibitor gefitinib similarly promotes the differentiation of AML cell lines and primary patient-derived AML blasts in vitro. Gefitinib induced differentiation based on morphologic assessment, nitro-blue tetrazolium reduction, cell-surface markers, genome-wide patterns of gene expression, and inhibition of proliferation at clinically achievable doses. Importantly, EGFR expression was not detected in AML cells, indicating that gefitinib functions through a previously unrecognized EGFR-independent mechanism. These studies indicate that clinical trials testing the efficacy of gefitinib in patients with AML are warranted.