Identification of a novel negative role of flagellin in regulating IL-10 production

Toll-like receptors (TLR) expressed by cells of the immune system play a central role in the generation of immune responses against pathogens. Following TLR ligation, both pro-inflammatory and anti-inflammatory mediators are produced in order to elicit an immune response that controls the microbial infection while limiting tissue damage. Among these mediators, the pro-inflammatory cytokine IL-12 and the anti-inflammatory cytokine IL-10 are known to play major roles. Here, we show that in vitro or in vivo stimulation with flagellin, the TLR5 ligand, does not result in IL-10 production. Furthermore, flagellin inhibits IL-10 production by other specific TLR ligands at the protein and mRNA levels while increasing IL-12p70 production. Several studies have linked the activation of extracellular signal-regulated kinases with IL-10 induction by TLR. We have observed that LPS-induced extracellular signal-regulated kinase activation was significantly decreased in flagellin-treated macrophages, suggesting that this pathway might play a role in the inhibition of IL-10 production observed in flagellin-treated macrophages. Flagellin-mediated IL-10 inhibition was not observed in cells that do not express TLR5, supporting that this effect is indeed TLR5-dependent. This study provides a new insight into the role of flagellin recognition by TLR5 in shaping the immune response elicited by flagellated microorganisms.     

Genetic variants in ZIC1, ZIC2, and ZIC3 are not major risk factors for neural tube defects in humans

Neural tube defects (NTD) are congenital malformations arising from incomplete neural tube closure during early embryogenesis. Most NTD in humans show complex inheritance patterns, with both genetic and environmental factors involved in the etiology of this malformation. More than 120 mouse models for human NTD exist. NTD have been observed in mice deficient for the Zic family genes, Zic1, Zic2, and Zic3. We performed mutation analysis in the human orthologs of these genes using DNA material from a large panel of NTD patients. In ZIC2 we identified a deletion of one codon that encodes an alanine residue located in the amino terminal alanine stretch of the protein. The deletion was present in one patient, but not in 364 controls. That may suggest a role-albeit small-of this variant in the etiology of NTD in humans. Transmission disequilibrium testing of a frequent polymorphism in the ZIC2 gene (1059C > T, H353H) in parent-spina bifida aperta child triads showed no association with NTD. One silent polymorphism (858G > A, V286V) of unknown significance was identified in ZIC3. Neither mutations nor polymorphisms were found in the coding region or flanking sequences of ZIC1. Our data indicate that ZIC1, ZIC2, and ZIC3 are not major risk factors for NTD in humans.     

Identification of a novel transglutaminase from the filarial parasite Brugia malayi and its role in growth and development.

Recently, we reported the presence of a putative transglutaminase in adult female worms of Brugia malayi [1]. The enzyme activity was shown to be essential for in utero growth and development of microfilariae. Here, we demonstrate that adult worms of B. malayi have a large amount of epsilon-(gamma-glutamyl)lysine isopeptide bonds, a product of physiologically active transglutaminase. A 25-kDa immunoreactive band detected in female worm extracts by a monospecific monoclonal antibody (CUB 7401) against guinea pig liver transglutaminase was associated with the enzymatic activity. Unlike the mammalian enzyme, the parasite enzyme did not require Ca2+ for its catalytic activity. Furthermore, in utero developing embryos, especially during early stages of development, contained very high amounts of this enzyme. Adult female worms contained several proteins that could serve as suitable substrates for the enzyme. Inhibition of the enzyme activity by an enzyme-specific pseudosubstrate, monodansylcadaverine, led to a time- and dose-dependent inhibition of microfilariae production and release by gravid female worms. The inhibition of microfilariae production was due to the inhibition of transglutaminase-catalyzed crosslinking of parasite proteins that in turn seemed to be essential for in utero growth and development of the embryos. The results suggest that transglutaminase-catalyzed reactions may play an important role during early development of embryos to mature microfilariae inside the adult female worms of filarial parasites.     

鋅指转录因子ZIC3在内脏异位中的研究现状及进展

内脏异位是由于左右非对称性发育异常所致,常与胸腹腔器官的异常偏侧化有关。心脏经常受累,且心脏受累的严重程度通常决定其预后效果。内脏异位患者有特征性的心血管畸形、内脏器官的异常排列以及中线结构发育畸形。在内脏异位患者中第一个被发现有突变的基因是编码锌指转录因子的ZIC3。很多研究证实,ZIC3突变可导致X连锁内脏异位,而且在孤立性先心病中也发现了ZIC3的突变。至今,在内脏异位患者中发现有13个ZIC3突变,其中包括无义突变、错义突变、沉默突变、移码突变以及易位突变等。然而,ZIC3基因在内脏异位,特别是伴复杂先心病中的致病机理仍不是很清楚。本文就ZIC3结构、作用、突变以及其在内脏异位伴先心病中的研究现状及存在的问题做一综述。     

Identification of a novel role for Drosophila MESR4 in lipid metabolism

Drosophila provides a powerful genetic model to analyze lipid metabolism. Drosophila has an adipose‐like organ called the fat body, which plays a crucial role in energy homeostasis. Here, we conducted a fat body‐specific misexpression screen to identify genes involved in lipid metabolism. We found that over‐expression of a nuclear protein with nine C₂H₂ type zinc‐finger motifs and a PHD‐finger, Misexpression suppressor of ras 4 (MESR4), reduces lipid accumulation in the fat body, whereas MESR4 knockdown increases it. We further show that MESR4 up‐regulates the expression of major lipases, which may account for the reduction in lipid storage in the fat body and the release of free fatty acids (FFAs) in the body. These results suggest that MESR4 acts as an important upstream regulator of energy homeostasis.     

Tim-3 and its role in regulating anti-tumor immunity

Immunotherapy is being increasingly recognized as a key therapeutic modality to treat cancer and represents one of the most exciting treatments for the disease. Fighting cancer with immunotherapy has revolutionized treatment for some patients and therapies targeting the immune checkpoint molecules such as CTLA-4 and PD-1 have achieved durable responses in melanoma, renal cancer, Hodgkin's diseases and lung cancer. However, the success rate of these treatments has been low and a large number of cancers, including colorectal cancer remain largely refractory to CTLA-4 and PD-1 blockade. This has provided impetus to identify other co-inhibitory receptors that could be exploited to enhance response rates of current immunotherapeutic agents and achieve responses to the cancers that are refectory to immunotherapy. Tim-3 is a co-inhibitory receptor that is expressed on IFN-g-producing T cells, FoxP3+ Treg cells and innate immune cells (macrophages and dendritic cells) where it has been shown to suppress their responses upon interaction with their ligand(s). Tim-3 has gained prominence as a potential candidate for cancer immunotherapy, where it has been shown that in vivo blockade of Tim-3 with other check-point inhibitors enhances anti-tumor immunity and suppresses tumor growth in several preclinical tumor models. This review discusses the recent findings on Tim-3, the role it plays in regulating immune responses in different cell types and the rationale for targeting Tim-3 for effective cancer immunotherapy.     

Identification and detection of the periostin gene in cardiac development

Periostin, a member of the fasciclin gene family, acts as a cell adhesion molecule through binding to cell surface integrins. Periostin expression has previously been shown to increase substantially following transforming growth factor beta (TGF-beta) and bone morphogenetic protein stimulation. As these molecules are indispensable for cardiac development, we sought to clone the chicken ortholog of periostin and evaluate its spatiotemporal expression pattern during heart morphogenesis. We show by Northern analysis, whole mount and section in situ hybridization experiments that periostin is predominantly expressed in the developing endothelium of the ventricular trabeculae as well as in the endothelium and mesenchyme of the outflow tract and atrioventricular endocardial cushions. Cardiac expression continues into fetal development where periostin is seen predominantly in the valve leaflets and supporting chordae tendinae.     

MicroRNAs在心脏发育和疾病中的作用

微小RNA(microRNA,miRNA)是一种长度为18-24核苷酸单链的内源性非编码小RNA,在进化过程中高度保守,通过与靶基因序列特异性相互作用在转录后水平调节基因表达,参与多种生物学过程。最初发现的内源性miRNA是lin-4和let-7,它们通过与靶mRNA的3'端非翻译区(untranslated region,UTR)相结合而发挥负性调节作用。以往的研究表明,miRNA主要参与器官形成、造血、细胞增殖与凋亡、肿瘤生成等生物学过程,而最近一些研究发现,miRNAs参与调节心脏的生长发育、机械重构和电重构过程。现就miRNA在心脏中作用的最新研究进展进行综述。     

Factors regulating vagal sensory development: potential role in obesities of developmental origin

Contributors to increased obesity in children may include perinatal under- or overnutrition. Humans and rodents raised under these conditions develop obesity, which like obesities of other etiologies has been associated with increased meal size. Since vagal sensory innervation of the gastrointestinal (GI) tract transmits satiation signals that regulate meal size, one mechanism through which abnormal perinatal nutrition could increase meal size is by altering vagal development, possibly by causing changes in the expression of factors that control it. Therefore, we have begun to characterize development of vagal innervation of the GI tract and the expression patterns and functions of the genes involved in this process. Important events in development of mouse vagal GI innervation occurred between midgestation and the second postnatal week, suggesting they could be vulnerable to effects of abnormal nutrition pre- or postnatally. One gene investigated was brain- derived neurotrophic factor (BDNF), which regulates survival of a subpopulation of vagal sensory neurons. BDNF was expressed in some developing stomach wall tissues innervated by vagal afferents. At birth, mice deficient in BDNF exhibited a 50% reduction of putative intraganglionic laminar ending mechanoreceptor precursors, and a 50% increase in axons that had exited fiber bundles. Additionally, BDNF was required for patterning of individual axons and fiber bundles in the antrum and differentiation of intramuscular array mechanoreceptors in the forestomach. It will be important to determine whether abnormal perinatal environments alter development of vagal sensory innervation of the GI tract, involving effects on expression of BDNF, or other factors regulating vagal development.     

Dandy-Walker malformation associated with heterozygous ZIC1 and ZIC4 deletion: Report of a new patient

We report on a female patient with Dandy-Walker malformation possibly caused by heterozygous loss of ZIC1 and ZIC4. The patient presented with mental retardation, epilepsy, and multiple congenital malformations including spina bifida, mild dysmorphic facial features including, thick eyebrows, broad nose, full lips, macroglossia, and hypoplasia of the cerebellar vermis with enlargement of the fourth ventricle on brain magnetic resonance imaging, which is consistent with Dandy-Walker malformation. A chromosome analysis showed interstitial deletion of chromosome 3q23-q25.1. Fluorescence in situ hybridization (FISH) and microarray-based genomic analysis revealed the heterozygous deletion of ZIC1 and ZIC4 loci on 3q24. Her facial features were not consistent with those observed in blepharophimosis-ptosis-epicanthus inversus syndrome (BPES) involving FOXL2 abnormality. Other deleted genes at 3q23-25.1 might contribute to the dysmorphic facial appearance. A milder phenotype as the Dandy-Walker malformation in our patient supports the idea that modifying loci/genes can influence the development of cerebellar malformation.     

The TGFβ type II receptor plays a critical role in the endothelial cells during cardiac development

TGFβ signalling is required for normal cardiac development. To investigate which cell types are involved, we used mice carrying a floxed Type II TGFβ receptor (Tgfbr2fl) allele and Cre‐lox genetics to deplete this receptor in different regions of the heart. The three target tissues and corresponding Cre transgenic lines were atrioventricular myocardium (using cGata6‐Cre), ventricular myocardium (using Mlc2v‐Cre), and vascular endothelium (using tamoxifen‐activated Cdh5(PAC)‐CreERT2). Spatio‐temporal Cre activity in each case was tracked via lacZ activation from the Rosa26R locus. Atrioventricular‐myocardial‐specific Tgfbr2 knockout (KO) embryos had short septal leaflets of the tricuspid valve, whereas ventricular myocardial‐specific KO embryos mainly exhibited a normal cardiac phenotype. Inactivation of Tgfbr2 in endothelial cells from E11.5 resulted in deficient ventricular septation, accompanied by haemorrhage from cerebral blood vessels. We conclude that TGFβ signalling through the Tgfbr2 receptor, in endothelial cells, plays an important role in cardiac development, and is essential for cerebral vascular integrity. Developmental Dynamics 239:2435–2442, 2010. © 2010 Wiley‐Liss, Inc.     

骨肉瘤相关新分子5D3Ag的研究

目的：自建的抗人骨肉瘤单克隆抗体mAb5D3经鉴定有很好的骨肉瘤细胞特异性，并且已利用单抗纯化出相应的骨肉瘤相关抗原5D3Ag纯品，研究目的是测定5D3Ag氨基酸序列，鉴定5D3Ag是否为一新发现的骨肉瘤相关抗原。方法：用SDS-PAGE法分析5D3Ag纯度和分子量，并做免疫印迹分析，用半干式转移法将5D3Ag转移到氨基酸测序膜上，测定其氨基酸序列，在蛋白质序列中比较分析，初步推断此抗原的性质及是否为新发现的骨肉瘤相关抗原。结果：5D3Ag经SDSPAGE分析达到了电泳纯，分子量为43kD；Western Blot分析显示5D3Ag可以与mAb5D3结合；5D3Ag N端10个氨基酸序列为：门冬酰胺、谷氨酰胺、甘氨酸、丝氨酸、组氨酸、甘氨酸、丝氨酸、谷氨酸、丙氨酸、谷氨酰胺(NQGSHGSEAQ)。结论：5D3Ag从其分子量来看与以往文献所报道的骨肉瘤相关抗原的分子量都不相同；氨基酸序列结果查询分析，与蛋白质库中已知的蛋白质同源性很低，由此推断5D3Ag可能是一种新的骨肉瘤相关抗原。