Alterations in the serum glycome due to metastatic prostate cancer

Glycomic profiles derived from human blood sera of 10 healthy males were compared to those from 24 prostate cancer patients. The profiles were acquired using MALDI-MS of permethylated N-glycans released from 10-microL sample aliquots. Quantitative permethylation was attained using solid-phase permethylation. Principal component analysis of the glycomic profiles revealed significant differences among the two sets, allowing their distinct clustering. The first principal component distinguished the 24 prostate cancer patients from the healthy individuals. It was determined that fucosylation of glycan structures is generally higher in cancer samples (ANOVA test p-value of 0.0006). Although more than 50 N-glycan structures were determined, 12 glycan structures, of which six were fucosylated, were significantly different between the two sample sets. Significant differences were confirmed through two independent statistical tests (ANOVA and ROC analyses). Ten of these structures had significantly higher relative intensities in the case of the cancer samples, while the other two were less abundant in the cancer samples. All 12 structures were statistically significant, as suggested by their very low ANOVA scores (<0.001) and ROC analysis, with area under the curve values close to 1 or 0. Accordingly, these structures can be considered as cancer-specific glycans and potential prostate cancer biomarkers. Therefore, serum glycomic profiling appears worthy of further investigation to define its role in cancer early detection and prognostication.     

Quantitative analysis of total serum glycome in human and mouse

Model mice are frequently used in drug discovery research. Knowledge of similarities and differences between the mouse and human glycomes is critical when model mice are used for the discovery of glycan‐related biomarkers and targets for therapeutic intervention. Since few comparative glycomic studies between human and mouse have been conducted, we performed a comprehensive comparison of the major classes of glycans in human and mouse sera using mass spectrometric and liquid chromatographic analyses. Up to 131 serum glycans, including N‐glycans, free oligosaccharides (fOSs), glycosaminoglycans, O‐glycans, and glycosphingolipid (GSL)‐glycans, were quantified. In both serum samples, N‐glycans were the most abundant in the total serum glycome, while fOSs were the least abundant. As expected, the diversity of sialic acid (i.e. Neu5Ac vs. Neu5Gc) was the major species difference between human and mouse in terms of N‐ and O‐glycosylation, while GSL‐glycomic profiles were completely different, even when the sialic acid diversity was taken into consideration. Furthermore, total serum glycomics of STAM mouse were unveiled as an initial step to identify novel biomarkers of liver diseases, with which we could identify several glycans with expression significantly increased or decreased expression.     

TLRs、慢性炎症与肿瘤

Toll样受体是新发现的先天性免疫的病原模式识别受体,在机体抵抗外来病原生物入侵上起关键的作用。除了抵抗外来病原生物入侵外, Toll样受体现在也被认为与某些自身免疫性疾病、肿瘤和某些病因不明的疾病的发病有关。慢性炎症被认为在不同层面促进某些肿瘤的发生和发展,其中之一就是慢性炎症通过免疫抑制导致免疫系统对肿瘤细胞无反应,这种免疫抑制也与 Toll样受体有关。     

IL-36 in chronic inflammation and cancer

IL-36 belongs to the IL-1 family of cytokines and activates target cells by binding to a specific cytokine receptor (IL-36R) followed by activation of intracellular regulators such as MAP kinases and NF-kappaB. Three subforms of IL-36, denoted IL-36alpha, IL-36beta and IL-36gamma, have been described that require N-terminal cleavage for activation. Functional studies have shown that IL-36 may activate a broad spectrum of immune and non-immune cells such as macrophages, T cells, keratinocytes and epithelial cells in an IL-1-independent fashion and thereby controls various inflammatory or oncogenic processes in the skin, the lung, the kidney, the liver and the intestine, respectively. Based on the presence of mutations of the IL-36RN in patients with generalized pustular psoriasis, successful clinical pilot trials with IL-36R blocking antibodies were conducted in these patients and further studies in patients with autoimmune or chronic inflammatory disorders such as inflammatory bowel diseases are under way. Collectively, these findings highlight a crucial regulatory role of IL-36 signaling in driving various inflammatory disorders that provide a rational basis for clinical targeting of this cytokine.     

Evaluation of serum exosomal LncRNA‐based biomarker panel for diagnosis and recurrence prediction of bladder cancer

Exosomes are small membrane vesicles released by many cells. These vesicles can mediate cellular communications by transmitting active molecules including long non‐coding RNAs (lncRNAs). In this study, our aim was to identify a panel of lncRNAs in serum exosomes for the diagnosis and recurrence prediction of bladder cancer (BC). The expressions of 11 candidate lncRNAs in exosome were investigated in training set (n = 200) and an independent validation set (n = 320) via quantitative real‐time PCR. A three‐lncRNA panel (PCAT‐1, UBC1 and SNHG16) was finally identified by multivariate logistic regression model to provide high diagnostic accuracy for BC with an area under the receiver‐operating characteristic curve (AUC) of 0.857 and 0.826 in training set and validation set, respectively, which was significantly higher than that of urine cytology. The corresponding AUCs of this panel for patients with Ta, T1 and T2‐T4 were 0.760, 0.827 and 0.878, respectively. In addition, Kaplan‐Meier analysis showed that non‐muscle‐invasive BC (NMIBC) patients with high UBC1 expression had significantly lower recurrence‐free survival (P = 0.01). Multivariate Cox analysis demonstrated that UBC1 was independently associated with tumour recurrence of NMIBC (P = 0.018). Our study suggested that lncRNAs in serum exosomes may serve as considerable diagnostic and prognostic biomarkers of BC.     

Genomics and epigenomics of the human glycome

The majority of all proteins are glycosylated and glycans have numerous important structural, functional and regulatory roles in various physiological processes. While structure of the polypeptide part of a glycoprotein is defined by the sequence of nucleotides in the corresponding gene, structure of a glycan part results from dynamic interactions between hundreds of genes, their protein products and environmental factors. The composition of the glycome attached to an individual protein, or to a complex mixture of proteins, like human plasma, is stable within an individual, but very variable between individuals. This variability stems from numerous common genetic polymorphisms reflecting in changes in the complex biosynthetic pathway of glycans, but also from the interaction with the environment. Environment can affect glycan biosynthesis at the level of substrate availability, regulation of enzyme activity and/or hormonal signals, but also through gene-environment interactions. Epigenetics provides a molecular basis how the environment can modify phenotype of an individual. The epigenetic information (DNA methylation pattern and histone code) is especially vulnerable to environmental effects in the early intrauterine and neo-natal development and many common late-onset diseases take root already at that time. The evidences showing the link between epigenetics and glycosylation are accumulating. Recent progress in high-throughput glycomics, genomics and epigenomics enabled first epidemiological and genome-wide association studies of the glycome, which are presented in this mini-review.     

Elevated serum monocyte chemoattractant protein-4 and chronic inflammation in overweight subjects

Objective: Chronic inflammation observed in obesity has been reported to be implicated in the development of atherosclerosis. We screened candidate chemokines that link chronic inflammation and obesity. Research Methods and Procedures: Japanese overweight (n = 39, BMI 28.7 ± 0.65 kg/m²) and normal‐weight (n = 24, BMI 22.3 ± 0.45 kg/m²) subjects were enrolled. Using antibody‐based protein microarray, spot intensities of monocyte chemoattractant protein (MCP)‐4, eotaxin, and eotaxin‐2 correlated with anthropometric parameters. We further measured serum concentration of these chemokines and mRNA levels in adipose tissues obtained from volunteers. Results: Serum MCP‐4 levels showed positive correlation with BMI (r = 0.318, p = 0.014), waist (r = 0.316, p = 0.018), and waist‐to‐hip ratio (WHR) (r = 0.264, p = 0.049). Furthermore, MCP‐4 correlated with homeostasis model assessment of insulin resistance (r = 0.392, p = 0.002), high‐sensitivity C‐reactive protein (hsCRP) (r = 0.350, p = 0.006). In step‐wise multiple regression analyses, hsCRP independently correlated with MCP‐4 levels. The expression of MCP‐4 mRNA in visceral adipose tissue positively correlates with BMI. Serum eotaxin levels correlate with BMI (r = 0.262, p = 0.045) and WHR (r = 0.383, p = 0.003). Serum eotaxin‐2 levels correlated with BMI (r = 0.464, p < 0.001), waist (r = 0.333, p = 0.017), and WHR (r = 0.278, p = 0.048). However, eotaxin and eotaxin‐2 levels did not show significant correlation with hsCRP. Discussion: Serum levels of MCP‐4, eotaxin, and eotaxin‐2, which belong to CC chemokine family and share CC chemokine receptor 3, correlated with BMI. These chemokines, especially MCP‐4, may be critical molecules that link obesity and chronic inflammation.     

国产血清半乳甘露聚糖检测试剂对侵袭性肺曲霉菌病的诊断价值评估

目的：评估国产血清半乳甘露聚糖（Galactomannan,GM）检测试剂对侵袭性肺曲霉菌病的诊断价值。方法根据血液病/恶性肿瘤患者侵袭性真菌病的诊断标准与治疗原则（第四次修订版）[1]收集临床确诊侵袭性肺曲霉菌病（inva-sive pulmonary aspergillosis,IPA）、临床诊断IPA、拟诊IPA、排除IPA四组病例。采用天津贻诺琦公司酶联免疫吸附法（ELISA）试剂检测纳入的86例患者血清标本的GM浓度,分析其敏感性、特异性、阳性预测值（PPV）、阴性预测值（NPV）。结果86例病例中,临床诊断27例、拟诊12例、排除47例。在3种不同的阳性判断标准下,敏感性：9444%、9630%、6296%;特异性：5625%、4576%、6441%;PPV：4474%、4483%、4474%;NPV：9643%、9643%、7917%。统计学分析证实标准1（即血清GM值〉095μg/L为阳性,〈075μg/L为阴性,075~095μg/L为灰区,未将灰区加入计算）在3种判断标准中最优,故选择其为最终判断标准。结论该血清GM检测试剂盒诊断性能较好,可以用于侵袭性肺曲霉菌病的辅助诊断。     

Evaluation of Verbascum species and harpagoside in models of acute and chronic inflammation

Verbascum species are widely used in folk medicine because of their broad range of biological activities. Harpagoside, an iridoid glycoside isolated from some Verbascum plants is known for its anti-inflammatory properties. In the present study, the effect of five extracts of Verbascum species and harpagosides were evaluated in mouse models of acute and chronic inflammation. The results demonstrate that Verbascum phoeniceum extract strongly inhibits COX-1 (60.2% inhibition vs PMA-stimulated cells) and COX-2 (44.8% inhibition) expression stimulated peritoneal macrophages resulting in reduced paw swelling in carrageenan-induced oedema (55.5% inhibition vs PBS-treated mice). Harpagoside ameliorated the development of zymosaninduced arthritis and reduced pathological changes in joints as shown by the decreased histological score for cell infiltration in synovial cavity (3.5±0.2 in vs 2.0±0.16), cartilage loss (2.5±0.3 vs 1.8±0.5) and bone resorption (2.4±0.2 vs 1.8±0.4). Molecular docking simulations of harpagoside suggest that it may function with increased specific affinity towards COX-1 than COX-2. The potential of harpagoside to be applied as an effective agent for treating joint-related disorders is discussed.     

Influence of chronic inflammation on the malignant phenotypes and the plasticity of colorectal cancer cells

Sporadic adenoma or adenocarcinoma is often detected during endoscopic surveillance of patients with ulcerative colitis (UC). However, it is occasionally difficult to distinguish these neoplasms from dysplasia or colitis-associated cancers because of the influence of inflammation. However, the influence of inflammation on sporadic neoplasms is not well characterised. To assess this influence, we established a long-term inflammation model of colon cancer cells by inflammatory stimulation with tumour necrosis factor-α, flagellin and interleukin-1β for 60 weeks. Then, the malignant phenotypes were evaluated using the MTS assay, Annexin V fluorescence assay, cell migration assay and sphere formation assay. The influence of P53 function on these phenotypes was assessed with a TP53 mutation model using the CRISPR/Cas9 system. A long-term inflammation model of LS174T cells was established for the first time with continuous inflammatory signalling. Chronic inflammation induced apoptosis and suppressed the proliferation and stemness of these cancer cells via the action of P53. It also enhanced the invasiveness of LS174T cells. Moreover, these phenotypic changes and changes in inflammatory signalling were recoverable after the removal of inflammatory stimuli, suggesting that colon cancer cells have higher plasticity than normal intestinal epithelial cells. In conclusion, our results suggest that sporadic neoplasms in patients with UC are affected by chronic inflammation but are not essentially altered.     

IL-6 signaling in autoimmunity, chronic inflammation and inflammation-associated cancer

IL-6 activates various cell types carrying the membrane bound IL-6R (classical IL-6 signaling) as well as IL-6R(-) gp130(+) cells via the soluble IL-6R (IL-6 trans-signaling). IL-6 signaling plays a pivotal role in controlling the differentiation and activation of T lymphocytes by inducing the Jak/STAT-3 and the Ras/Erk/C/EBP pathways. In particular, IL-6 modulates the resistance of T cells against apoptosis, induces activation of T helper cells and controls the balance between regulatory T cells and Th17 cells. Importantly, recent findings suggest that blockade of IL-6 signaling is effective in treating experimental models of autoimmune and chronic inflammatory diseases such as inflammatory bowel diseases, diabetes, multiple sclerosis, asthma and rheumatoid arthritis as well as models of inflammation-associated cancer. Thus, anti-IL-6/anti-IL-6R strategies emerge as promising novel approaches for therapy of inflammatory diseases in humans. In this review article, we discuss the latest findings on the role of IL-6 in experimental models of autoimmunity and cancer, as well as clinical perspectives.     

The development of retrosynthetic glycan libraries to profile and classify the human serum N‐linked glycome

Annotation of the human serum N‐linked glycome is a formidable challenge but is necessary for disease marker discovery. A new theoretical glycan library was constructed and proposed to provide all possible glycan compositions in serum. It was developed based on established glycobiology and retrosynthetic state‐transition networks. We find that at least 331 compositions are possible in the serum N‐linked glycome. By pairing the theoretical glycan mass library with a high mass accuracy and high‐resolution MS, human serum glycans were effectively profiled. Correct isotopic envelope deconvolution to monoisotopic masses and the high mass accuracy instruments drastically reduced the amount of false composition assignments. The high throughput capacity enabled by this library permitted the rapid glycan profiling of large control populations. With the use of the library, a human serum glycan mass profile was developed from 46 healthy individuals. This paper presents a theoretical N‐linked glycan mass library that was used for accurate high‐throughput human serum glycan profiling. Rapid methods for evaluating a patient's glycome are instrumental for studying glycan‐based markers.     

The mannose receptor ligands and the macrophage glycome

A unique glycan-binding protein expressed in macrophages and some types of other immune cells is the mannose receptor (MR, CD206). It is an endocytic, transmembrane protein with multiple glycan-binding domains and different specificities in binding glycans. The mannose receptor is important as it has major roles in diverse biological processes, including regulation of circulating levels of reproductive hormones, homeostasis, innate immunity, and infections. These different functions involve the recognition of a wide range of glycans, and their nature is currently under intense study. But the mannose receptor is just one of many glycan-binding proteins expressed in macrophages, leading to an interest in the potential relationship between the macrophage glycome and how it may regulate cognate glycan-binding protein activities. This review focuses primarily on the mannose receptor and its carbohydrate ligands, as well as macrophages and their glycomes.     

Delineating diseases by IMS-MS profiling of serum N-linked glycans

Altered branching and aberrant expression of N-linked glycans is known to be associated with disease states such as cancer. However, the complexity of determining such variations hinders the development of specific glycomic approaches for assessing disease states. Here, we examine a combination of ion mobility spectrometry (IMS) and mass spectrometry (MS) measurements, with principal component analysis (PCA) for characterizing serum N-linked glycans from 81 individuals: 28 with cirrhosis of the liver, 25 with liver cancer, and 28 apparently healthy. Supervised PCA of combined ion-mobility profiles for several, to as many as 10 different mass-to-charge ratios for glycan ions, improves the delineation of diseased states. This extends an earlier study [J. Proteome Res.2008, 7, 1109-1117] of isomers associated with a single glycan (S(1)H(5)N(4)) in which PCA analysis of the IMS profiles appeared to differentiate the liver cancer group from the other samples. Although performed on a limited number of test subjects, the combination of IMS-MS for different combinations of ions and multivariate PCA analysis shows promise for characterizing disease states.     

N-linked glycosylation of the liver cancer biomarker GP73

The association between elevated circulating levels of GP73 (and fucosylated GP73 in particular) and hepatocellular carcinoma suggests that a thorough analysis of the extent of GP73 glycosylation is warranted. Detailed analysis of the glycosylation patterns of such low abundance proteins are hampered by technical difficulties. Using conventional lectin affinity chromatography, we have established that three quarters of the GP73 secreted from a cell line derived from HCC is fucosylated. Using mass spectrometry, we have established that at least two of three potential sites of N-linked glycosylation are occupied on most molecules of GP73 secreted from cultured hepatoma cells. Furthermore, the oligosaccharides added to recombinant GP73 resemble those present in the bulk of secreted protein, mostly bi-antennary with core fucose, with a smaller fraction of tri- and tetra-antennary structures. The frequency of fucosylation observed on the recombinant protein agrees well with the pattern of lectin binding of the endogenous secreted protein. Finally, we have developed a method to interrogate the glycans added to either the near full length protein or at a particular sequon, providing proof of concept that a small peptide embedded in a heterologous context can preserve both fucosylation and a high level of branching of oligosaccharides added.     

Insights into miRNA regulation of the human glycome

Glycosylation is an intricate process requiring the coordinated action of multiple proteins, including glycosyltransferases, glycosidases, sugar nucleotide transporters and trafficking proteins. Work by several groups points to a role for microRNA (miRNA) in controlling the levels of specific glycosyltransferases involved in cancer, neural migration and osteoblast formation. Recent work in our laboratory suggests that miRNA are a principal regulator of the glycome, translating genomic information into the glycocode through tuning of enzyme levels. Herein we overlay predicted miRNA regulation of glycosylation related genes (glycogenes) onto maps of the common N-linked and O-linked glycan biosynthetic pathways to identify key regulatory nodes of the glycome. Our analysis provides insights into glycan regulation and suggests that at the regulatory level, glycogenes are non-redundant.     

IL-17+ regulatory T cells in the microenvironments of chronic inflammation and cancer

Foxp3(+)CD4(+) regulatory T (Treg) cells inhibit immune responses and temper inflammation. IL-17(+)CD4(+) T (Th17) cells mediate inflammation of autoimmune diseases. A small population of IL-17(+)Foxp3(+)CD4(+) T cells has been observed in peripheral blood in healthy human beings. However, the biology of IL-17(+)Foxp3(+)CD4(+) T cells remains poorly understood in humans. We investigated their phenotype, cytokine profile, generation, and pathological relevance in patients with ulcerative colitis. We observed that high levels of IL-17(+)Foxp3(+)CD4(+) T cells were selectively accumulated in the colitic microenvironment and associated colon carcinoma. The phenotype and cytokine profile of IL-17(+)Foxp3(+)CD4(+) T cells was overlapping with Th17 and Treg cells. Myeloid APCs, IL-2, and TGF-β are essential for their induction from memory CCR6(+) T cells or Treg cells. IL-17(+)Foxp3(+)CD4(+) T cells functionally suppressed T cell activation and stimulated inflammatory cytokine production in the colitic tissues. Our data indicate that IL-17(+)Foxp3(+) cells may be "inflammatory" Treg cells in the pathological microenvironments. These cells may contribute to the pathogenesis of ulcerative colitis through inducing inflammatory cytokines and inhibiting local T cell immunity, and in turn may mechanistically link human chronic inflammation to tumor development. Our data therefore challenge commonly held beliefs of the anti-inflammatory role of Treg cells and suggest a more complex Treg cell biology, at least in the context of human chronic inflammation and associated carcinoma.     

肥胖与慢性炎症

肥胖及其相关的代谢类疾病严重影响人类的健康,而肥胖诱导的慢性炎症是胰岛素抵抗和代谢综合症发病的关键因素.脂肪组织慢性炎症发生的机制及其与代谢综合症的关系已经成为全球瞩目的研究热点.慢性炎症的特征主要包括脂肪组织中促炎细胞因子表达量增加,抗炎细胞因子表达量降低以及大量巨噬细胞浸润.鉴于肥胖及其相关代谢综合症对人类健康的巨大危害,现对慢性炎症的发生机制,肥胖和慢性炎症之间的关系,脂肪组织炎症中巨噬细胞浸润以及和信号传导通路进行综述.     

Hepatic catabolism of serum amyloid A during an acute phase response and chronic inflammation

Degradation of serum amyloid A (SAA) was studied in isolated perfused livers of mice treated with either a single injection of casein to induce an acute phase response or with 14 daily casein injections to maintain chronic inflammation. Littermates administered sterile saline served as controls. Radioiodinated SAA and apolipoprotein A-I, reconstituted with high-density lipoproteins in vivo, were studied in parallel. Degradation was monitored by appearance of acid-soluble radioactivity in the perfusate. Induction of an acute phase response reduced hepatic catabolism of SAA by 14% (from 8.6 +/- 1.2% to 7.4 +/- 1.1%/g liver in 3 hr, P less than 0.05, n = 16). The acute phase response had no effect on apolipoprotein A-I degradation or bile production. Livers from animals receiving 14 daily injections of casein were 31% less active than control livers at degrading SAA (8.1 +/- 1.6%/g/3 hr for treated group vs. 11.7 +/- 2.3%/g/3 hr for control group, P less than 0.025). Apolipoprotein A-I degradation was decreased but differences were not statistically significant and bile production was the same in both treatment groups. However, livers from treated animals were larger (mean weight 1.8 g) than those from controls (1.5 g) (P less than 0.05), although amyloid fibrils were not detected by Congo red stain. The size of the degradation products was analyzed by column chromatography. Elution profiles of perfusates from livers of chronically inflamed animals contained a peak corresponding to the molecular weight of amyloid A which was not present in perfusates from control liver. We conclude that hepatic catabolism of SAA is decreased both early and late in an inflammatory response and intermediate degradation products corresponding in size to amyloid A are released into the circulation following prolonged inflammation.