Pathways to immunity: temporal dynamics of the bumblebee (Bombus terrestris) immune response against a trypanosomal gut parasite

Immune response dynamics in insects from natural host-parasite associations are poorly understood, despite accumulating evidence of ecological immune phenomena in these systems. Using a gene discovery approach, we have identified genes relating to signalling, enzymatic processes and respiration that were up-regulated in the bumblebee, Bombus terrestris, during infection with the trypanosomatid parasite, Crithidia bombi. In addition, we have mapped dynamic changes in the temporal expression of these genes and three candidate antimicrobial peptide (AMP) immune genes, Abaecin, Defensin and Hymenoptaecin, from 1 to 24 h after C. bombi infection. We show that dynamic changes in expression occur for individual genes at distinct phases of the immune response to C. bombi that correspond to early, intermediate and late stages of infection.     

Induction of {beta}-defensin resistance in the oral anaerobe Porphyromonas gingivalis

Induction of resistance of oral anaerobes to the effects of human beta-defensin 1 (hbetaD-1) to hbetaD-4 was investigated by pretreating cells with either sublethal levels of defensins or environmental factors, followed by a challenge with lethal levels of defensins. Cultures of Porphyromonas gingivalis were (i) pretreated with defensins at 1 ng/ml, (ii) heated to 42 degrees C (heat stress), (iii) exposed to normal atmosphere (oxidative stress), or (iv) exposed to 1 mM hydrogen peroxide (peroxide stress). Samples (10 microl) were distributed among the wells of sterile 384-well plates containing hbetaD-1 to -4 (100 microg/ml). Plates were incubated at 37 degrees C for 36 h in an anaerobe chamber. Growth inhibition was determined by a system that measures the total nucleic acid of a sample with a DNA binding dye. The MICs of the four defensins for P. gingivalis were 3 to 12 microg/ml. We found that sublethal levels of the defensins and heat and peroxide stress, but not oxidative stress, induced resistance to 100 microg of defensin per ml in P. gingivalis. Resistance induced by sublethal levels of hbetaD-2 lasted 90 min, and the resistance induced by each defensin was effective against the other three. Multiple strains exposed to hbetaD-2 all evidenced resistance induction. Defensin resistance is vital to the pathogenic potential of several human pathogens. This is the first report describing the induction of defensin resistance in the oral periodontal pathogen P. gingivalis. Such resistance may have an effect on the ability of oral pathogens to persist in the mouth and to withstand innate human immunity.     

Identification and molecular characterization of defensin gene from the ant Formica aquilonia

The effectors of the insect immune system are antimicrobial peptides. With the aim of studying the evolution of immune system genes, we identified a gene encoding the antimicrobial peptide defensin from a social insect, the wood ant Formica aquilonia. In this article we report the identification and characterization of this gene. We also compare the ant defensin gene structure to that previously obtained from two other hymenopteran species, the honeybee, Apis mellifera, and the bumblebee, Bombus ignitus. The ant defensin gene structure differs from both of these bee defensins with respect to the number and length of introns and exons.     

The expression patterns of peritoneal defensins.

BACKGROUND: Local defense mechanisms are important for the integrity of the peritoneum, but few details are known about the expression patterns of antimicrobial proteins such as human defensin in normal and damaged peritoneum. METHODS: Part A: The expression of different defensins in normal (n = 12), inflamed (n = 5), and metastatic peritoneum (n = 4) and in cultured human peritoneal mesothelial cells was analyzed using mRNA and immunohistochemistry. Part B: Using immunohistochemistry the expression of different defensins was analyzed in different subgroups: healthy controls (n = 25), patients with chronic appendicitis (n = 25) or acute appendicitis (n = 10), and end-stage renal disease patients (n = 25, with 15 on peritoneal dialysis). RESULTS: Part A: Human neutrophil peptides (HNP) 1 and 3 and human beta-defensins (HBD) 1 to 3 mRNA were detected in peritoneal specimens. In addition, HNP1,3, HBD1, HBD2, and HBD3 proteins were detected using immunohistochemistry. Part B: HBD1 showed a constitutive expression in mesothelium, while HBD2 and HNP1,3 were associated with inflammation. Decreased expressions of HNP1,3 were observed in end-stage renal disease patients and in patients on peritoneal dialysis. CONCLUSIONS: For the first time, the expression patterns of defensins in normal and damaged peritoneum have been described. The reduced expression of some defensins in end-stage renal disease is of potential clinical interest against the background of the frequent infective complications seen in peritoneal dialysis.     

Role of defensins in inflammatory lung disease

The human airways are protected from invading micro-organisms by the highly efficient innate immune system. Antimicrobial peptides that are produced by inflammatory cells and airway epithelial cells are key elements in this innate immune system. A major subgroup of the antimicrobial peptides is the family of defensins - small non-enzymatic and cationic peptides. Besides their extensively studied role in antimicrobial defense, recent studies have demonstrated that defensins are also able to modulate inflammatory responses, to stimulate adaptive immunity and contribute to tissue repair. In line with these observations, increased defensin levels were observed in inflammatory lung diseases, such as cystic fibrosis (CF), diffuse panbroncheolitis (DPB), idiopathic pulmonary fibrosis (IPF) and acute respiratory distress syndrome (ARDS), and in infectious diseases. In the past decade much has been learnt about the activity of defensins and there is abundant evidence for their presence in human inflammatory lung disease. Future studies are required to elucidate their role in the pathogenesis of these diseases.     

Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor MprF is based on modification of membrane lipids with l-lysine

Defensins, antimicrobial peptides of the innate immune system, protect human mucosal epithelia and skin against microbial infections and are produced in large amounts by neutrophils. The bacterial pathogen Staphylococcus aureus is insensitive to defensins by virtue of an unknown resistance mechanism. We describe a novel staphylococcal gene, mprF, which determines resistance to several host defense peptides such as defensins and protegrins. An mprF mutant strain was killed considerably faster by human neutrophils and exhibited attenuated virulence in mice, indicating a key role for defensin resistance in the pathogenicity of S. aureus. Analysis of membrane lipids demonstrated that the mprF mutant no longer modifies phosphatidylglycerol with l-lysine. As this unusual modification leads to a reduced negative charge of the membrane surface, MprF-mediated peptide resistance is most likely based on repulsion of the cationic peptides. Accordingly, inactivation of mprF led to increased binding of antimicrobial peptides by the bacteria. MprF has no similarity with genes of known function, but related genes were identified in the genomes of several pathogens including Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Enterococcus faecalis. MprF thus constitutes a novel virulence factor, which may be of general relevance for bacterial pathogens and represents a new target for attacking multidrug resistant bacteria.     

Role of defensins in inflammatory lung disease

The human airways are protected from invading micro-organisms by the highly efficient innate immune system. Antimicrobial peptides that are produced by inflammatory cells and airway epithelial cells are key elements in this innate immune system. A major subgroup of the antimicrobial peptides is the family of defensins--small non-enzymatic and cationic peptides. Besides their extensively studied role in antimicrobial defense, recent studies have demonstrated that defensins are also able to modulate inflammatory responses, to stimulate adaptive immunity and contribute to tissue repair. In line with these observations, increased defensin levels were observed in inflammatory lung diseases, such as cystic fibrosis (CF), diffuse panbroncheolitis (DPB), idiopathic pulmonary fibrosis (IPF) and acute respiratory distress syndrome (ARDS), and in infectious diseases. In the past decade much has been learnt about the activity of defensins and there is abundant evidence for their presence in human inflammatory lung disease. Future studies are required to elucidate their role in the pathogenesis of these diseases.     

Human autoantibody to defensin: disease association with hyperreactive onchocerciasis (sowda)

Chronic hyperreactive onchodermatitis (sowda) is a severe form of onchocerciasis observed in a subset of individuals infected with the filarial nematode Onchocerca volvulus. SDS-PAGE and immunoblot analyses of O. volvulus adult worm extracts were used to characterize the antigens of the marked antibody response of sowda patients. One 2.5-kD antigen was recognized by sera from all 35(100%) sowda patients that were studied. In comparison, only 7 of 44 (16%) patients with generalized onchocerciasis and 11 of 21 (52%) of exposed individuals with no microfilariae in skin snips and no signs of disease showed reactivity to this antigen. Microfilaricidal treatment of sowda patients with improvement of the clinical status was associated with a decrease or disappearance of antibodies to the 2.5-kD antigen. Amino acid sequencing of the antigen indicated identity to human defensins 1- 3 of neutrophils. Defensin was demonstrated by immunohistochemical staining in onchocercal nodules on the surface of adult filariae and in the surrounding tissue. A similar staining pattern was observed for other proteins present in neutrophils such as myeloperoxidase, elastase, and the L-1 protein complex (MRP 8/MRP 14), indicating that neutrophils, macrophages, and their proteins predominate in the environment adjacent to the worms. These results demonstrate an association between the presence of autoantibodies to defensins and an infectious disease of known etiology. The association with a particular form of onchocerciasis, sowda, suggests a link between formation of autoantibodies to defensin and enhanced immune reactivity towards the parasite.     

Convergence of IL-1β and VDR Activation Pathways in Human TLR2/1-Induced Antimicrobial Responses

Antimicrobial effector mechanisms are central to the function of the innate immune response in host defense against microbial pathogens. In humans, activation of Toll-like receptor 2/1 (TLR2/1) on monocytes induces a vitamin D dependent antimicrobial activity against intracellular mycobacteria. Here, we report that TLR activation of monocytes triggers induction of the defensin beta 4 gene (DEFB4), requiring convergence of the IL-1β and vitamin D receptor (VDR) pathways. TLR2/1 activation triggered IL-1β activity, involving the upregulation of both IL-1β and IL-1 receptor, and downregulation of the IL-1 receptor antagonist. TLR2/1L induction of IL-1β was required for upregulation of DEFB4, but not cathelicidin, whereas VDR activation was required for expression of both antimicrobial genes. The differential requirements for induction of DEFB4 and cathelicidin were reflected by differences in their respective promoter regions; the DEFB4 promoter had one vitamin D response element (VDRE) and two NF-κB sites, whereas the cathelicidin promoter had three VDREs and no NF-κB sites. Transfection of NF-κB into primary monocytes synergized with 1,25D3 in the induction of DEFB4 expression. Knockdown of either DEFB4 or cathelicidin in primary monocytes resulted in the loss of TLR2/1-mediated antimicrobial activity against intracellular mycobacteria. Therefore, these data identify a novel mechanism of host defense requiring the induction of IL-1β in synergy with vitamin D activation, for the TLR-induced antimicrobial pathway against an intracellular pathogen.     

Gene organization of a novel defensin of Ixodes ricinus: first annotation of an intron/exon structure in a hard tick defensin gene and first evidence of the occurrence of two isoforms of one member of the arthropod defensin family

Antimicrobial peptides (defensins) are effectors of the immune system. Herein, we describe a novel Ixodes ricinus defensin gene(s), analyse its structure and compare it with other known antimicrobial peptides from different tick species. For the first time, an intron/exon structure is discovered in a hard-tick defensin gene. The intron/exon genomic organization of the gene is similar to the organization in Ornithodoros moubata, but not to that of the intronless defensins of Dermacentor variabilis and Ixodes scapularis. The analysis of the deduced amino acid sequences of different recombinants from the I. ricinus cDNA library reveals the presence of two defensin isoforms with three amino acid substitutions. Whether or not these substitutions affect the biological properties of the peptides is currently unknown. The expression of the defensin gene is strongly induced in the tick midgut after infection with Borrelia burgdorferi.     

Antimicrobial peptides and the skin

In recent years, hundreds of naturally occurring peptide antibiotics have been discovered based on their ability to inhibit the growth of microbial pathogens. These antimicrobial peptides (AMPs) participate in the innate immune response by providing a rapid first-line defence against infection. This review discusses the biology and clinical relevance of the two major families of AMPs, cathelicidins and defensins, with emphasis on their function in mammalian skin and their association with skin pathology. Current evidence shows that cathelicidins and defensins act as both natural antibiotics and as signalling molecules that activate host cell processes involved in immune defence and tissue repair. Alterations in the expression pattern of AMPs have been associated with a variety of pathological processes. Ongoing and future studies are likely to implicate AMPs in several unexplained human inflammatory disorders and to provide novel therapeutic approaches for the treatment of these diseases.     

Antimicrobial peptides and the skin

In recent years, hundreds of naturally occurring peptide antibiotics have been discovered based on their ability to inhibit the growth of microbial pathogens. These antimicrobial peptides (AMPs) participate in the innate immune response by providing a rapid first-line defence against infection. This review discusses the biology and clinical relevance of the two major families of AMPs, cathelicidins and defensins, with emphasis on their function in mammalian skin and their association with skin pathology. Current evidence shows that cathelicidins and defensins act as both natural antibiotics and as signalling molecules that activate host cell processes involved in immune defence and tissue repair. Alterations in the expression pattern of AMPs have been associated with a variety of pathological processes. Ongoing and future studies are likely to implicate AMPs in several unexplained human inflammatory disorders and to provide novel therapeutic approaches for the treatment of these diseases.     

应用寡聚核苷酸芯片检测白血病与其同胞供者的白血病基因表达差异

为了应用寡聚核苷酸基因表达谱芯片研究9对白血病患者／同胞供受者对之间的基因表达谱差异以识别主要的疾病相关基因，将163条白血病／肿瘤发病相关基因组群列入芯片设计，合成寡核苷酸探针，用点样仪点片制成基因芯片。提取病初白血病患者及其供者的骨髓／外周血标本或其相对应的健康同胞供者经G-CSF动员后并经CS-3000细胞分离机采集的浓集的9份外周血造血干细胞的总RNA；分别逆转录并进行寡核苷酸探针杂交。结果表明：与其相应的正常供者配对比较，发现4例ALL患者中存在6条显著差异表达基因，其中上调表达基因5条(RIZ，STK-1，T—cell leukemia／lymphoma 1A，Cbp／p300，Opl8)，下调表达1条(hematopoietic proteoglycan core protein)；5例AML-M4和AML-M，患者中亦存在7条显著差异表达基因，其中上调表达6条(STAT5B，ligand p62 for the Lck SH2，CST3，LTC4S，myeloid leukemia factor 2，epb72)，下调表达1条(CCR5)。结论：选择有高度遗传关联的兄弟姐妹供受者对作为差异研究比照对象，利用寡聚核苷酸基因芯片技术进行疾病基因的筛查，筛查出了13条主要异常基因；进一步确认了上述基因在白血病分子发病机制中的关键性作用。     

2型糖尿病家族史风险通路识别及功能分析

目的: 探讨2型糖尿病家族史与胰岛素抵抗间的关系,采用信息学方法对从基因表达库（Gene Expression Omnibus, GEO）获取的具有2型糖尿病家族史但自身血糖正常的研究对象进行相关基因表达谱分析,识别2型糖尿病疾病风险通路,并对关键基因产物进行功能分析。方法: 对美国国立生物技术信息中心（National Center for Biotechnology Information,NCBI）的综合性基因表达与杂交阵列数据库中26名有2型糖尿病家族史但自身血糖正常的研究对象和15名无糖尿病家族史的血糖正常的人群进行数据分析（GSE25462）,运用Bergman微小模型技术结合静脉糖耐量试验评估胰岛素敏感指数,并对骨骼肌细胞基因表达谱芯片采用生物信息学方法（基因功能富集分析方法、风险通路识别方法等）进行数据分析,进而在分子层面进行2型糖尿病致病机制研究。结果: 有2型糖尿病家族史但自身血糖正常的研究对象其胰岛素敏感性下降41%（P<0.05）。通过差异表达分析,共发现202个基因表达在家族史阳性组与家族史阴性组间的差异有统计学意义,同时鉴定了富集差异显著的5条通路,分别是多能干细胞的造血功能、万能干细胞的造血功能、JAK1/3在c-细胞活素信号通路的作用、TOB在T细胞信号通路中的抗增殖作用、B细胞的发育。结论: 有2型糖尿病家族史但自身血糖正常的研究对象存在胰岛素抵抗,2型糖尿病的致病机制与造血干细胞功能及免疫状态有重要联系。