结核分枝杆菌感染过程中宿主抗菌肽LL-37免疫调控功能的研究进展

抗菌肽是先天免疫系统的重要组成部分,LL-37是已知唯一在人体中表达的抗菌肽。LL-37除了具有抗结核活性和免疫调节等生物学功能外,还可能是活动性结核的重要标识。本文着重就结核分枝杆菌感染调控LL-37表达机制、LL-37在结核分枝杆菌感染过程中的免疫防御以及免疫调节功能等方面进行阐述。另外,讨论了LL-37在结核病诊断、治疗中的应用潜力,以期为结核病预防和控制工作提供新的思路。     

人β-防御素-2与肺部疾病的研究进展

人β-防御素-2(human β-defensin 2,HBD-2)是一类在肺部诱导性表达的抗菌肽,在呼吸系统的防御中发挥着重要作用.本文就HBD-2的分子结构、生物学活性、表达调控及其在呼吸系统疾病中的研究进展进行综述.     

防御素在抗结核分枝杆菌感染中的作用机制研究进展

防御素是广泛分布于生物界的一类富含半胱氨酸的内源性阳离子抗菌肽,在抵抗病原微生物入侵及调节机体免疫功能等方面发挥重要作用。研究发现,防御素对细菌、真菌或病毒等均具有杀灭作用,其在抗结核分枝杆菌感染中也展现出突出优势。防御素独特的抗菌机理可以破坏结核分枝杆菌的细胞壁、细胞膜等生物学结构,从而抑制结核分枝杆菌。本文对防御素的分子结构、生物学作用、抗结核作用及机制予以综述。     

LL-37抗菌肽重组及其治疗实验性内毒素血症的研究

目的重组抗菌肽LL-37并探讨其治疗内毒素血症的疗效。方法提取人肺腺上皮SPC-A-1细胞总RNA,RT-PCR扩增出LL-37肽cDNA,构建pGEX-1λT-LL-37重组质粒并转染大肠杆菌JM109,培养转染细菌并诱导表达融合蛋白,裂解细菌,亲和层析出融合蛋白,裂解融合蛋白生成LL-37肽,高压液相色谱法提纯LL-37;建立BALB／C小鼠内毒素血症模型并观察LL-37的疗效。结果重组肽核苷序列5’-端比天然序列多4个密码子,N-端多4个氨基酸残基。小、大剂量LL-37治疗组（Ⅰ组、Ⅱ组）及内毒素血症组（Ⅲ组）6h存活率分别为62．5％、87．5％、0;Ⅰ、Ⅱ组平均存活时间和最长存活时间均长于Ⅲ组;Ⅰ、Ⅱ组24h后小鼠存活率有差异,量效关系明显。结论重组抗菌肽LL-37可用于内毒素血症的治疗。     

β-防御素-2与Toll样受体关系的研究进展

β-防御素-2是固有免疫应答的重要组成成分,是-类可诱导性的抗菌肽,抗菌谱广,对革兰阳性菌、革兰阴性菌、真菌、分枝杆菌、非典型病原体和包膜病毒都具有-定的抵抗作用。关于β防御素-2的生成机制虽然尚未完全阐明,但大量研究表明与上皮细胞表达的Toll样受体密切相关,本文就β防御素-2与Toll样受体关系的研究进展作一综述。     

补充维生素D3治疗对成人支气管扩张症患者肺功能、LCQ评分、抗菌肽LL-37和IL-8水平的影响

目的探讨补充维生素D3治疗对成人支气管扩张症患者肺功能、生命质量、抗菌肽LL-37和IL-8水平的影响。方法选择2016年2月至2017年2月在我院住院治疗的支气管扩张症且血清25(OH)D≤30 ng/mL的患者97例,随机分为观察组49例和对照组48例。对照组患者均给予支气管扩张症常规治疗,观察组患者在对照组治疗的基础上补充维生素D3治疗,800 IU/d,连续治疗观察6个月。比较两组患者治疗前后的肺功能,莱塞斯特咳嗽生命质量问卷(LCQ)评分,血清25(OH)D、抗菌肽LL-37和IL-8水平。结果治疗前后,两组患者的FVC、FEV1%水平比较差异无统计学意义(P0.05);治疗6个月后,观察组患者LCQ评分明显提高且显著高于对照组(P0.05);观察组患者的血清25(OH)D、LL-37水平明显升高且显著高于对照组,血清IL-8水平明显降低且显著低于对照组,差异有统计学意义(P0.05)。观察组患者血清25(OH)D变化值与血清LL-37变化值呈弱正相关(r=0.662,P=0.001)。结论补充维生素D3治疗,可提高支气管扩张症患者血清25(OH)D水平,但不能明显改善其肺功能;可减少促炎介质的产生;随着血25(OH)D水平的升高,可以上调抗菌肽LL-37的表达,增强免疫应答反应,有助于患者防御病原微生物和促炎性细胞因子的攻击。     

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。     

抗菌肽：人类健康和疾病中的重要角色

抗菌肽是生物体防御系统产生的一类多肽。越来越多的研究表明抗菌肽不仅能够直接抑制和杀灭病原微生物,同时具有多种免疫调节活性,在宿主的天然和适应性免疫中起着重要的作用,因而具有广阔的应用前景。它们的表达水平与人类的许多疾病密切相关。本文对两类主要的人源抗菌肽：防御素和cathelicidin的结构特性、表达和生物活性及其与疾病的关系作一综述。     

抗菌肽LL-37抗寄生虫活性研究进展

由原虫、蠕虫感染导致的寄生虫病在全球范围内广泛流行,尤其是在热带和亚热带地区,对儿童和成人生命健康均造成威胁。抗寄生虫药的长期使用导致寄生虫对药物敏感性下降甚至出现耐药性。研究表明抗菌肽可抑制寄生虫生长发育,具有潜在抗寄生虫价值,其中LL-37作为组织蛋白酶抑制素(cathelicidin)家族中唯一的人源抗菌肽被广泛研究。本文综述了LL-37抗寄生虫作用的研究进展,并对其作为抗寄生虫药物的候选资源的应用前景进行了探讨。     

人β-防御素-2在肺部炎性疾病的研究进展

人类为抵抗外界微生物侵袭,逐步形成一套完善的天然免疫和获得性免疫系统.人防御素作为重要的天然免疫成分,近10年成为研究热点.各脏器黏膜表面分泌的β-防御素是机体主要的防御屏障.而人β-防御素-2在感染和炎症诱导下在肺部进行高水平表达,成为研究防御素和肺部感染性和非感染性疾病的临床课题.     

Enterohemorrhagic and enteropathogenic Escherichia coli evolved different strategies to resist antimicrobial peptides

Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) are enteric human pathogens that colonize the large and small intestines, respectively. To establish infection EHEC and EPEC must overcome innate host defenses, such as antimicrobial peptides (AMPs) produced by the intestinal epithelium. Gram-negative pathogens have evolved different mechanisms to resist AMPs, including outer-membrane proteases that degrade AMPs. We showed that the protease OmpT degrades the human AMP LL-37 more rapidly in EHEC than in EPEC. Promoter-swap experiments showed that this is due to differences in the promoters of the two genes, leading to greater ompT expression and subsequently greater levels of OmpT in EHEC. Here, we propose that the different ompT expression in EHEC and EPEC reflects the varying levels of LL-37 throughout the human intestinal tract. These data suggest that EHEC and EPEC adapted to their specific niches by developing distinct AMP-specific resistance mechanisms.     

Enterohemorrhagic and enteropathogenic Escherichia coli evolved different strategies to resist antimicrobial peptides

Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) are enteric human pathogens that colonize the large and small intestines, respectively. To establish infection EHEC and EPEC must overcome innate host defenses, such as antimicrobial peptides (AMPs) produced by the intestinal epithelium. Gram-negative pathogens have evolved different mechanisms to resist AMPs, including outer-membrane proteases that degrade AMPs. We showed that the protease OmpT degrades the human AMP LL-37 more rapidly in EHEC than in EPEC. Promoter-swap experiments showed that this is due to differences in the promoters of the two genes, leading to greater ompT expression and subsequently greater levels of OmpT in EHEC. Here, we propose that the different ompT expression in EHEC and EPEC reflects the varying levels of LL-37 throughout the human intestinal tract. These data suggest that EHEC and EPEC adapted to their specific niches by developing distinct AMP-specific resistance mechanisms.     

Antiviral Effect of hBD-3 and LL-37 during Human Primary Keratinocyte Infection with West Nile Virus

West Nile virus (WNV) is an emerging flavivirus transmitted through mosquito bites and responsible for a wide range of clinical manifestations. Following their inoculation within the skin, flaviviruses replicate in keratinocytes of the epidermis, inducing an innate immune response including the production of antimicrobial peptides (AMPs). Among them, the cathelicidin LL-37 and the human beta-defensin (hBD)-3 are known for their antimicrobial and immunomodulatory properties. We assessed their role during WNV infection of human primary keratinocytes. LL-37 reduced the viral load in the supernatant of infected keratinocytes and of the titer of a viral inoculum incubated in the presence of the peptide, suggesting a direct antiviral effect of this AMP. Conversely, WNV replication was not inhibited by hBD-3. The two peptides then demonstrated immunomodulatory properties whether in the context of keratinocyte stimulation by poly(I:C) or infection by WNV, but not alone. This study demonstrates the immunostimulatory properties of these two skin AMPs at the initial site of WNV replication and the ability of LL-37 to directly inactivate West Nile viral infectious particles. The results provide new information on the multiple functions of these two peptides and underline the potential of AMPs as new antiviral strategies in the fight against flaviviral infections.     

Expression of LL-37 by human gastric epithelial cells as a potential host defense mechanism against Helicobacter pylori

BACKGROUND & AIMS : LL-37/human cationic antimicrobial peptide 18 (hCAP18) is a human cathelicidin with broad-spectrum antimicrobial, lipopolysaccharide binding, and chemotactic activities. This study examined the role of LL-37/hCAP18 in gastric innate immune defense by characterizing its constitutive and regulated expression by human gastric mucosa and its bactericidal activity against the gastric pathogen Helicobacter pylori. METHODS : LL-37/hCAP18 messenger RNA expression in normal and H. pylori -infected gastric mucosa and gastric epithelial cells was determined by in situ hybridization, real-time polymerase chain reaction, immunostaining, and immunoblot analysis. Bactericidal activity was measured by using a colony-forming unit assay. RESULTS : LL-37/hCAP18 messenger RNA and protein were expressed in a distinct distribution by surface epithelial cells as well as chief and parietal cells in the fundic glands of normal gastric mucosa. LL-37/hCAP18 was significantly increased in the epithelium and gastric secretions of H. pylori -infected patients, but not in individuals with non-H. pylori -induced gastric inflammation. Infection of cultured gastric epithelial cells with a wild-type but not an isogenic Delta cagE mutant strain of H. pylori increased LL-37/hCAP18 expression, indicating that H. pylori -induced regulation of LL-37/hCAP18 production required an intact type IV secretion system. LL-37, the C-terminal peptide of LL-37/hCAP18, alone or in synergy with human beta-defensin 1, was bactericidal for several H. pylori strains. CONCLUSIONS : These data indicate that H. pylori up-regulates production of LL-37/hCAP18 by gastric epithelium and suggest this cathelicidin contributes to determining the balance between host mucosal defense and H. pylori survival mechanisms that govern chronic infection with this gastric pathogen.     

Marked Reduction of LL-37/hCAP-18, an Antimicrobial Peptide,in Patients with Acute Myeloid Leukemia

We detected LL-37/hCAP-18 expression in the peripheral blood smears of 50 healthy donors and 143 patients with various hematological diseases. Compared with that in the healthy donors, expression of the protein in the neutrophils was significantly lower in patients with acute myeloid leukemia (AML), especially those with infection, but no significant difference was detected in messenger RNA level. We did not detect increased LL-37/hCAP-18 protein expression in U937 cells treated with lipopolysaccharide or Staphylococcus aureus Cowan strain. Furthermore, LL-37/hCAP-18 protein production was not restored in differentiated myeloid cell lines NB4 or HL-60 induced by all-trans retinoic acid. LL-37/hCAP-18 has been shown to play a role in host defense, and its deficiency in AML may be one of the explanations for susceptibility to infection among these patients.     

The peptide antibiotic LL-37/hCAP-18 is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface

The airway surface is an important host defense against pulmonary infection. Secretion of proteins with antimicrobial activity from epithelial cells onto the airway surface represents an important component of this innate immune system. Defensins are the best characterized epithelial-derived peptide antibiotics. A member of another family of peptide antibiotics called cathelicidins recently was identified from human bone marrow. We show in this paper that this human peptide named LL-37/hCAP-18 also may play a role in innate immunity of the human lung. In situ hybridization localized high levels of LL-37/hCAP-18 RNA to surface epithelial cells of the conducting airway as well as serous and mucous cells of the submucosal glands. LL-37/hCAP-18 peptide with antimicrobial activity was partially purified from airway surface fluid from human lung and a human bronchial xenograft model. The synthetic peptide LL-37 demonstrated antibiotic activity against a number of Gram-negative and Gram-positive organisms including Pseudomonas aeruginosa; bacterial killing of LL-37 was sensitive to NaCl and was synergistic with lactoferrin and lysozyme. In summary, we show that LL-37/hCAP-18 is a peptide with broad antimicrobial activity that is secreted onto the airway surface from epithelial cells of the human lung.     

Real-time attack on single Escherichia coli cells by the human antimicrobial peptide LL-37

Natural antimicrobial peptides (AMPs) provide prototypes for the design of unconventional antimicrobial agents. Existing bulk assays measure AMP activity but do not provide details of the growth-halting mechanism. We use fluorescence microscopy to directly observe the attack of the human antimicrobial peptide LL-37 on single Escherichia coli cells in real time. Our findings strongly suggest that disruption of the cytoplasmic membrane is not the growth-halting mechanism. At 8 μM, LL-37 binding saturates the outer membrane (OM) within 1 min. Translocation across the OM and access to the periplasmic space (5-25 min later) correlates in time with the halting of growth. Septating cells are attacked more readily than nonseptating cells. The halting of growth may occur because of LL-37 interference with cell wall biogenesis. Only well after growth halts does the peptide permeabilize the cytoplasmic membrane to GFP and the small dye Sytox Green. The assay enables dissection of antimicrobial design criteria into two parts: translocation across the OM and the subsequent halting of growth.     

Antibacterial components in bronchoalveolar lavage fluid from healthy individuals and sarcoidosis patients.

Antibacterial peptides and proteins are an integral part of the epithelial defense barrier that provides immediate protection against bacterial invasion. In humans, alpha-defensins are mainly bactericidal effectors in circulating granulocytes, beta-defensin-1 is synthesized in epithelial cells, and LL-37 is produced in granulocytes but is also induced in skin epithelia during inflammation. To investigate the importance of these defense effectors in disease, we analyzed bronchoalveolar lavage fluid (BALF) for bactericidal activity. Antibacterial activity was found in BALF material from healthy individuals and sarcoidosis patients, with enhanced activity in BALF from the patients. The activity was present as several antibacterial components, of which we have so far characterized LL-37, lysozyme, alpha-defensins, and antileukoprotease. In addition, the antibacterial peptide LL-37 was located in alveolar macrophages, bronchial epithelial cells, and bronchial glands, suggesting that it has a defensive role in airway mucosa. In conclusion, the airway epithelium is protected by a complex antibacterial defense system. This is activated in sarcoidosis, and may explain why these patients seldom develop severe respiratory tract infections.     

Increased levels of antimicrobial peptides in tracheal aspirates of newborn infants during infection

Pneumonia and systemic infection are common in premature infants. The antimicrobial peptides human beta-defensin 1 and 2 (hBD-1 and hBD-2) and the cathelicidin LL-37/hCAP-18 are effector molecules of the innate respiratory immune system. It is unknown whether these host defense substances are produced in the respiratory tract of newborns. Concentrations of these peptides were determined in tracheal aspirates of mechanically ventilated newborn infants. All three antimicrobial peptides could be detected in airway lining fluid with equivalent levels in term and preterm newborns. Concentrations of antimicrobial peptides correlated with each other and with levels of interleukin-8 and tumor necrosis factor-alpha in the bronchoalveolar lavage fluid. Pulmonary or systemic infections were associated with significantly increased concentrations of LL-37, hBD-1, and hBD-2. Western blotting detected mature peptides in the lavage fluid. In conclusion, mucosal antimicrobial peptides are present in lung secretions of premature and mature newborns. The molecules are upregulated in response to infection and inflammation and probably represent effector molecules of the respiratory defense system.