Interleukin 6 Receptor Is an Independent Prognostic Factor and a Potential Therapeutic Target of Ovarian Cancer

Ovarian cancer remains the most lethal gynecologic cancer and new targeted molecular therapies against this miserable disease continue to be challenging. In this study, we analyzed the expressional patterns of Interleukin-6 (IL-6) and its receptor (IL-6R) expression in ovarian cancer tissues, evaluated the impact of these expressions on clinical outcomes of patients, and found that a high-level of IL-6R expression but not IL-6 expression in cancer cells is an independent prognostic factor. In in vitro analyses using ovarian cell lines, while six (RMUG-S, RMG-1, OVISE, A2780, SKOV3ip1 and OVCAR-3) of seven overexpressed IL-6R compared with a primary normal ovarian surface epithelium, only two (RMG-1, OVISE) of seven cell lines overexpressed IL-6, suggesting that IL-6/IL-6R signaling exerts in a paracrine manner in certain types of ovarian cancer cells. Ovarian cancer ascites were collected from patients, and we found that primary CD11b⁺CD14⁺ cells, which were predominantly M2-polarized macrophages, are the major source of IL-6 production in an ovarian cancer microenvironment. When CD11b⁺CD14⁺ cells were co-cultured with cancer cells, both the invasion and the proliferation of cancer cells were robustly promoted and these promotions were almost completely inhibited by pretreatment with anti-IL-6R antibody (tocilizumab). The data presented herein suggest a rationale for anti-IL-6/IL-6R therapy to suppress the peritoneal spread of ovarian cancer, and represent evidence of the therapeutic potential of anti-IL-6R therapy for ovarian cancer treatment.     

A DPP-4 Inhibitor Suppresses Fibrosis and Inflammation on Experimental Autoimmune Myocarditis in Mice

Myocarditis is a critical inflammatory disorder which causes life-threatening conditions. No specific or effective treatment has been established. DPP-4 inhibitors have salutary effects not only on type 2 diabetes but also on certain cardiovascular diseases. However, the role of a DPP-4 inhibitor on myocarditis has not been investigated. To clarify the effects of a DPP-4 inhibitor on myocarditis, we used an experimental autoimmune myocarditis (EAM) model in Balb/c mice. EAM mice were assigned to the following groups: EAM mice group treated with a DPP-4 inhibitor (linagliptin) (n = 19) and those untreated (n = 22). Pathological analysis revealed that the myocardial fibrosis area ratio in the treated group was significantly lower than in the untreated group. RT-PCR analysis demonstrated that the levels of mRNA expression of IL-2, TNF-α, IL-1β and IL-6 were significantly lower in the treated group than in the untreated group. Lymphocyte proliferation assay showed that treatment with the DPP-4 inhibitor had no effect on antigen-induced spleen cell proliferation. Administration of the DPP-4 inhibitor remarkably suppressed cardiac fibrosis and reduced inflammatory cytokine gene expression in EAM mice. Thus, the agents present in DPP-4 inhibitors may be useful to treat and/or prevent clinical myocarditis.     

Luminescence enhancement of the catalytic 19 kDa protein (KAZ) of Oplophorus luciferase by three amino acid substitutions

To characterize the luminescence properties of nanoKAZ, a 16 amino acid substituted mutant of the catalytic 19 kDa protein (KAZ) of Oplophorus luciferase, the effects of each mutated amino acid were investigated by site-specific mutagenesis. All 16 single substituted KAZ mutants were expressed in Escherichia coli cells and their secretory expressions in CHO-K1 cells were also examined using the signal peptide sequence of Gaussia luciferase. Luminescence activity of KAZ was significantly enhanced by single amino acid substitutions at V44I, A54I, or Y138I. Further, the triple mutant KAZ-V44I/A54I/Y138I, named eKAZ, was prepared and these substitutions synergistically enhanced luminescence activity, showing 66-fold higher activity than wild-KAZ and also 7-fold higher activity than nanoKAZ using coelenterazine as a substrate. Substrate specificity of eKAZ for C2- and/or C6-modified coelenterazine analogues was different from that of nanoKAZ, indicating that three amino acid substitutions may be responsible for the substrate recognition of coelenterazine to increase luminescence activity. In contrast, these substitutions did not stimulate protein secretion from CHO-K1 cells, suggesting that the folded-protein structure of KAZ might be different from that of nanoKAZ.     

Preparation of chitin/cellulose composite gels and films with ionic liquids

In this study, we performed preparation and characterizations of the chitin/cellulose composite gels and films using the two ionic liquids, 1-allyl-3-methylimidazolium bromide and 1-butyl-3-methylimidazolium chloride. First, chitin and cellulose were dissolved in each appropriate ionic liquid. Then, the two liquids were mixed in the desired ratios at 100 °C to give the homogeneous mixtures. The gels were obtained by standing the mixtures for 4 days. On the other hand, the films were obtained by casting the mixtures on glass plates, followed by soaking in water and drying. The obtained gels and films were characterized by XRD and TGA measurements. The mechanical properties of the gels and films were evaluated under compressive and tensile modes, respectively.     

Expression of semaphorin 3A in the rat corneal epithelium during wound healing

The neural guidance protein semaphorin 3A (Sema3A) is expressed in corneal epithelial cells of the adult rat. We have now further investigated the localization of Sema3A in the normal rat corneal epithelium as well as changes in its expression pattern during wound healing after central corneal epithelial debridement. The expression pattern of Sema3A was compared with that of the tight-junction protein zonula occludens-1 (ZO-1), the gap-junction protein connexin43 (Cx43), or the cell proliferation marker Ki67. Immunofluorescence analysis revealed that Sema3A was present predominantly in the membrane of basal and wing cells of the intact corneal epithelium. The expression of Sema3A at the basal side of basal cells was increased in the peripheral epithelium compared with that in the central region. Sema3A was detected in all layers at the leading edge of the migrating corneal epithelium at 6 h after central epithelial debridement. The expression of Sema3A was markedly up-regulated in the basal and lateral membranes of columnar basal cells apparent in the thickened, newly healed epithelium at 1 day after debridement, but it had largely returned to the normal pattern at 3 days after debridement. The expression of ZO-1 was restricted to superficial epithelial cells and remained mostly unchanged during the wound healing process. The expression of Cx43 in basal cells was down-regulated at the leading edge of the migrating epithelium but was stable in the remaining portion of the epithelium. Ki67 was not detected in basal cells of the central epithelium at 1 day after epithelial debridement, when Sema3A was prominently expressed. Immunoblot analysis showed that the abundance of Sema3A in the central cornea was increased 1 day after epithelial debridement, whereas that of ZO-1 or Cx43 remained largely unchanged. This increase in Sema3A expression was accompanied by up-regulation of the Sema3A coreceptor neuropilin-1. Our observations have thus shown that the expression of Sema3A is increased markedly in basal cells of the newly healed corneal epithelium, and that this up-regulation of Sema3A is not associated with cell proliferation. They further suggest that Sema3A might play a role in the regulation of corneal epithelial wound healing.     

Sialic Acid-focused Quantitative Mouse Serum Glycoproteomics by Multiple Reaction Monitoring Assay

Despite increasing importance of protein glycosylation, most of the large-scale glycoproteomics have been limited to profiling the sites of N-glycosylation. However, in-depth knowledge of protein glycosylation to uncover functions and their clinical applications requires quantitative glycoproteomics eliciting both peptide and glycan sequences concurrently. Here we describe a novel strategy for the multiplexed quantitative mouse serum glycoproteomics based on a specific chemical ligation, namely, reverse glycoblotting technique, focusing sialic acids and multiple reaction monitoring (MRM). LC-MS/MS analysis of de-glycosylated peptides identified 270 mouse serum peptides (95 glycoproteins) as sialylated glycopeptides, of which 67 glycopeptides were fully characterized by MS/MS analyses in a straightforward manner. We revealed the importance of a fragment ion containing innermost N-acetylglucosamine (GlcNAc) residue as MRM transitions regardless the sequence of the peptides. Versatility of the reverse glycoblotting-assisted MRM assays was demonstrated by quantitative comparison of 25 targeted glycopeptides from 16 proteins between mice with homo and hetero types of diabetes disease model.Clinical proteomics focusing on the identification and validation of biomarkers and the discovery of proteins as therapeutic targets is an emerging and highly important area of proteomics. Biomarkers are measurable indicators of a specific biological state (particularly one relevant to the risk of contraction) and the presence or the stage of disease, and are thus expected to be useful for the prediction, detection, and diagnosis of disease as well as to follow the efficacy, toxicology, and side effects of drug treatment, and to provide new functional insights into biological processes.At present, proteomics methods based on mass spectrometry (MS) have emerged as the preferred strategy for discovery of diagnostic, prognostic, and therapeutic protein biomarkers. Most biomarker discovery studies use unbiased, “identified-based” approaches that rely on high performance mass spectrometers and extensive sample processing. Semiquantitative comparisons of protein relative abundance between disease and control patient samples are used to identify proteins that are differentially expressed and, thus, to populate lists of potential biomarkers. De novo proteomics discovery experiments often result in tens to hundreds of candidate biomarkers that must be subsequently verified in serum. However, despite the large numbers of putative biomarkers, only a small number of them are passed through the development and validation process into clinical practice, and their rate of introduction is declining. The first non-standard abbreviation (MS above is standard) must be footnoted the same as the abbreviation footnote, and MRM must be the first abbreviation in the list because it is the one footnoted. After that the order does not matter.Targeted proteomics using multiple reaction monitoring (MRM)1 is emerging as a technology that complements the discovery capabilities of shotgun strategies as well as an alternative powerful novel MS-based approach to measure a series of candidate biomarkers (1–7). Therefore, MRM is expected to provide a powerful high throughput platform for biomarker validation, although clinical validation of novel biomarkers has been traditionally relying on immunoassays (8, 9). MRM exploits the unique capabilities of triple quadrupoles (QQQ) MS for quantitative analysis. In MRM, the first and the third quadrupoles act as filters to specifically select predefined m/z values corresponding to the peptide precursor ion and specific fragment ion of the peptide, whereas the second quadrupole serves as collision cell. Several such transitions (precursor/fragment ion pairs) are monitored over time, yielding a set of chromatographic traces with retention time and signal intensity for a specific transition as coordinates. These measurements have been multiplexed to provide 30 or more specific assays in one run. Such methods are slowly gaining acceptance in the clinical laboratory for the routine measurement of endogenous metabolites (10) (e.g. in screening newborns for a panel of inborn errors of metabolism) some drugs (11) (e.g. immunosuppressants), and the component analysis of sugars (12).One of the profound challenges in clinical proteomics is the need to handle highly complex biological mixtures. This complexity presents unique analytical challenges that are further magnified with the use of clinical serum/plasma samples to search for novel biomarkers of human disease. The serum proteome is composed of tens of thousands of unique proteins, of which concentrations may exceed 10 orders of magnitude. Protein glycosylation, one of the most common post-translational modifications, generates tremendous diversity, complexity, and heterogeneity of gene products. It changes the biological and physical properties of proteins, which include functions as signals or ligands to control their distribution, antigenicity, metabolic fate, stability, and solubility. Protein glycosylation, in particular by N-linked glycans, is prevalent in proteins destined for extracellular environments. These include proteins on the extracellular side of the plasma membrane, secreted proteins, and proteins contained in body fluids (such as blood serum, cerebrospinal fluid, urine, breast milk, saliva, lung lavage fluid, or pancreatic juice). Considering that such body fluids are most easily accessible for diagnostic and therapeutic purposes, it is not surprising that many clinical biomarkers and therapeutic targets are glycoproteins. These include, for example, cancer antigen 125 (CA125) in ovarian cancer, human epidermal growth factor receptor 2 (Her2/neu) in breast cancer, and prostate-specific antigen (PSA) in prostate cancer. In addition, changes in the extent of glycosylation and the structure of N-glycans or O-glycans attached to proteins on the cell surface and in body fluids have been shown to correlate with cancer and other disease states, highlighting the clinical importance of this modification as an indicator or effector of pathologic mechanisms (13–16). Thus, clinical proteomic platforms should have capability to provide protein glycosylation information as well as sufficient analytical depth to reliably detect and quantify specific proteins with sufficient accuracy and throughput.To improve the detection limits to the required sensitivities, one needs to dramatically reduce the complexity of the sera samples. For focused glycoproteomics, several techniques using lectins or antibodies enabling the large-scale identification of glycoproteins have recently been developed (17–19). Notably, Zhang et al. reported a method for the selective isolation of peptides based on chemical oxidation of the carbohydrate moiety and subsequent conjugation to a solid support using hydrazide chemistry (20–26). However, it is not possible to provide any structural information about N-glycans because the MS analysis is performed on peptides of which N-glycans are removed preferentially by treating with peptide N-glycanase (PNGase). In 2007, we developed a method for rapid enrichment analysis of peptides bearing sialylated N-glycans on the MALDI-TOF-MS platform (27). The method involves highly selective oxidation of sialic acid residues of glycopeptides to elaborate terminal aldehyde group and subsequent enrichment by chemical ligation with a polymer reagent, namely, reverse glycoblotting technique inspired from an original concept of glycoblotting method (28). This method, in principle, is capable identifying both glycan and peptide sequences concurrently. Recently, Nilsson et al. reported that glycopeptides from human cerebrospinal fluid can be enriched on the basis of the same principle as the reverse glycoblotting protocol, and captured glycopeptides were analyzed with ESI FT-ICR MS (29). Because it is well known that sialic acids play important roles in various biological processes including cell differentiation, immune response, and oncogenesis (30–34), our attention has been directed toward feasibility of the reverse glycoblotting technique in quantitative analysis of the specific glycopeptides carrying sialic acid(s) by combining with multiplexed MRM-based MS.     

Changes of Gap and Tight Junctions during Differentiation of Human Nasal Epithelial Cells Using Primary Human Nasal Epithelial Cells and Primary Human Nasal Fibroblast Cells in a Noncontact Coculture System

The epithelium of upper respiratory tissues such as nasal mucosa forms a continuous barrier to a wide variety of exogenous antigens. The epithelial barrier function is regulated in large part by the intercellular junctions, referred to as gap and tight junctions. However, changes of gap and tight junctions during differentiation of human nasal epithelial (HNE) cells are still unclear. In the present study, to investigate changes of gap and tight junctions during differentiation of HNE cells in vitro, we used primary human HNE cells cocultured with primary human nasal fibroblast (HNF) cells in a noncontact system. In HNE cells cocultured with HNF cells for 2 weeks, numerous elongated cilia-like structures were observed compared to those without HNF cells. In the coculture, downregulation of Cx26 and upregulation of Cx30.3 and Cx31 were observed together with extensive gap junctional intercellular communication. Furthermore, expression of the tight junction proteins claudin-1, claudin-4, occludin and ZO-2 was increased. These results suggest that switching in expression of connexins and induction of tight junction proteins may be closely associated with differentiation of HNE cells in vitro and that differentiation of HNE cells requires unknown soluble factors secreted from HNF cells.     

Structural comparison of fucosylated and nonfucosylated Fc fragments of human immunoglobulin G1

Removal of the fucose residue from the oligosaccharides attached to Asn297 of human immunoglobulin G1 (IgG1) results in a significant enhancement of antibody-dependent cellular cytotoxicity (ADCC) via improved IgG1 binding to Fcgamma receptor IIIa. To provide structural insight into the mechanisms of affinity enhancement, we determined the crystal structure of the nonfucosylated Fc fragment and compared it with that of fucosylated Fc. The overall conformations of the fucosylated and nonfucosylated Fc fragments were similar except for hydration mode around Tyr296. Stable-isotope-assisted NMR analyses confirmed the similarity of the overall structures between fucosylated and nonfucosylated Fc fragments in solution. These data suggest that the glycoform-dependent ADCC enhancement is attributed to a subtle conformational alteration in a limited region of IgG1-Fc. Furthermore, the electron density maps revealed that the traces between Asp280 and Asn297 of our fucosylated and nonfucosylated Fc crystals were both different from that in previously reported isomorphous Fc crystals.     

Pathogen recognition by Toll-like receptor 2 activates Weibel-Palade body exocytosis in human aortic endothelial cells

The endothelial cell-specific granule Weibel-Palade body releases vasoactive substances capable of modulating vascular inflammation. Although innate recognition of pathogens by Toll-like receptors (TLRs) is thought to play a crucial role in promotion of inflammatory responses, the molecular basis for early-phase responses of endothelial cells to bacterial pathogens has not fully been understood. We here report that human aortic endothelial cells respond to bacterial lipoteichoic acid (LTA) and synthetic bacterial lipopeptides, but not lipopolysaccharide or peptidoglycan, to induce Weibel-Palade body exocytosis, accompanied by release or externalization of the storage components von Willebrand factor and P-selectin. LTA could activate rapid Weibel-Palade body exocytosis through a TLR2- and MyD88-dependent mechanism without de novo protein synthesis. This process was at least mediated through MyD88-dependent phosphorylation and activation of phospholipase Cgamma. Moreover, LTA activated interleukin-1 receptor-associated kinase-1-dependent delayed exocytosis with de novo protein synthesis and phospholipase Cgamma-dependent activation of the NF-kappaB pathway. Increased TLR2 expression by transfection or interferon-gamma treatment increased TLR2-mediated Weibel-Palade body exocytosis, whereas reduced TLR2 expression under laminar flow decreased the response. Thus, we propose a novel role for TLR2 in induction of a primary proinflammatory event in aortic endothelial cells through Weibel-Palade body exocytosis, which may be an important step for linking innate recognition of bacterial pathogens to vascular inflammation.