Search

From the reasons that canine coronavirus (CCV) grows more efficiently than feline coronavirus in a cell culture and they are mutually related in their antigenicities, an enzyme-linked immunosorbent assay (ELISA) using CCV-infected feline kidney (CRFK) cells as substrate antigens was developed for detection of anti-coronavirus antibodies in cats. It was indispensable for generating coronavirus-specific ELISA antibody activities that the sample was applied to the mock-infected, normal CRFK cells in parallel with the CCV-infected cells and then the optical density values given by the mock-infected cell antigen were subtracted from those given by the virus-infected cell antigen. On the basis of ELISA antibody titers obtained in sera from the cats experimentally infected with CCV and from the spontaneous feline infectious peritonitis (FIP) cases, the ELISA described in the present study was found to be applicable as a simple and easy serologic test which was able to detect anti-coronavirus antibodies as efficiently as the indirect immunofluorescence assay with homologous FIP virus.

Osteopontin plays an important role in the development and perpetuation of rheumatoid arthritis (RA). Antibodies targeting osteopontin have shown promising therapeutic benefits against this disease. We have previously reported a novel anti-RA monoclonal antibody, namely, 23C3, and shown it capable of alleviating the symptoms of RA in a murine collagen-induced arthritis model, restoring the cytokine production profile in joint tissues, and reducing T-cell recall responses to collagen type II. We describe here the crystal structure of 23C3 in complex with its epitope peptide. Analyses of the complex structure reveal the molecular mechanism of osteopontin recognition by 23C3. The peptide folds into two tandem β-turns, and two key residues of the peptide are identified to be critical for the recognition by 23C3: TrpP43 is deeply embedded into a hydrophobic pocket formed by AlaL34, TyrL36, LeuL46, TyrL49, PheL91, and MetH102 and therefore has extensive hydrophobic interactions with 23C3, while AspP47 has a network of hydrophilic interactions with residues ArgH50, ArgH52, SerH53, and AsnH56 of the antibody. Besides the complementarity-determining region loops, the framework region L2 of 23C3 is also shown to interact with the epitope peptide, which is not common in the antibody–antigen interactions and thus could be exploited in the engineering of 23C3. These results not only provide valuable information for further improvement of 23C3 such as chimerization or humanization for its therapeutic application, but also reveal the features of this specific epitope of osteopontin that may be useful for the development of new antibody drugs against RA.

Rat small intestinal goblet cell mucins reacting with monoclonal antibody HCM31 increase significantly during regeneration from experimental mucosal damage and at the period of expulsion of parasitic nematode, Nippostrongylus brasiliensis (N.b). The reduction in reactivity of HCM31 with mucin upon neuraminidase treatment, suggested that HCM31 recognizes sialylated oligosaccharide on mucin. HCM31-reactive sialomucins are therefore considered to play an important role in the physiological and pathological changes in the gastrointestinal mucosa. To determine the epitope for HCM31, oligosaccharide–alditols reacted with HCM31 were obtained from the small intestinal mucins of N.b-infected rats and purified by ion-exchange chromatography followed by normal-phase HPLC. Two HCM31-reactive oligosaccharide-alditols were obtained. Analyses using tandem mass spectrometry and NMR spectroscopy showed that these oligosaccharides were core 4 mucin-type oligosaccharides having a common tetrasaccharide sequence, NeuAcα2-3(GalNAcβ1-4)Galβ1-4GlcNAcβ- (Sda blood group antigen). These structures were not found in the small intestinal mucin oligosaccharides from uninfected rats. This epitope specificity of HCM31 was also confirmed using previously established anti-GM2 and anti-Sda antibodies. Taken together, these results strongly suggest that HCM31 specifically recognizes mucin-type oligosaccharides with the Sda tetrasaccharide sequence. Immunohistochemical examination of human gastrointestinal tracts showed that HCM31 site-specifically stained the goblet cells in normal sigmoid colon and normal rectum, but the goblet cells stained with HCM31 were reduced in the corresponding cancer tissues. HCM31 seems to be useful for diagnosis of colonic cancer and for examining the function of secretory-type mucin with Sda antigen., Graphical abstract Highlights ▸ We analyzed the epitope for the monoclonal antibody HCM31. ▸ HCM31 recognizes core 4 oligosaccharides with the Sda antigen obtained from small intestinal mucins of N. brasiliensis-infected rats. ▸ We propose that the Sda tetrasaccharide sequence, NeuAcα2-3(GalNAcβ1-4)Galβ1-4GlcNAcβ-, is the epitope for HCM31. ▸ Goblet cells stained with HCM31 decrease with malignant change in human colon tissues. ▸ HCM31 might be useful for the diagnosis of colonic cancer and for examining the function of mucin with Sda tetrasaccharide.

Within the research and development environment, higher throughput, parallelized protein purification is required for numerous activities, from small scale purification of monoclonal antibodies (mAbs) and antibody fragments for in vitro and in vivo assays to process development and optimization for manufacturing. Here, we describe specific applications and associated workflows of the Protein Maker liquid handling system utilized in both of these contexts. To meet the requirements for various in vitro assays, for the identification and validation of new therapeutic targets, small quantities of large numbers of purified antibodies or antibody fragments are often required. Reducing host cell proteins (HCP) levels following capture with Protein A by evaluating various wash buffers is an example of how parallelized protein purification can be leveraged to improve a process development outcome. Stability testing under various conditions of in-process intermediates, as an example, the mAb product from a clarified harvest, requires parallelized protein purification to generate concurrent samples for downstream assays. We have found that the Protein Maker can be successfully utilized for small-to-mid scale platform purification or for process development applications to generate the necessary purified protein samples. The ability to purify and buffer exchange up to 24 samples in parallel offers a significant reduction in time and cost per sample compared to serial purification using a traditional FPLC system. By combining the Protein Maker purification system with a TECAN Freedom EVO liquid handler for automated buffer exchange we have created a new, integrated platform for a variety of protein purification and process development applications.

Background Neuromyelitis optica (NMO) is an inflammatory disease of the central nervous system that predominantly affects the optic nerves and spinal cord. Although NMO has long been considered a subtype of multiple sclerosis (MS), the effects of interferon-β treatment are different between NMO and MS. Recent findings of NMO-IgG suggest that NMO could be a distinct disease rather than a subtype of MS. However, the underlying molecular mechanism of NMO pathology remains poorly understood. Methods OPN in the cerebrospinal fluid and brain of patients with NMO and with MS, as well as of patients with other neurologic disease/idiopathic other neurologic disease was examined using Western blotting, ELISA, immunohistochemistry and Boyden chamber. Results Here we show that osteopontin is significantly increased in the cerebrospinal fluid of NMO patients compared with MS patients. Immunohistochemical analyses revealed that osteopontin was markedly elevated in the cerebral white matter of NMO patients and produced by astrocytes, neurons, and oligodendroglia as well as infiltrating macrophages. We also demonstrate that the interaction of the cerebrospinal fluid osteopontin in NMO patients with integrin αvβ3 promoted macrophage chemotaxis by activating phosphoinositide 3-kinase and MEK1/2 signaling pathways. Conclusion These results indicate that osteopontin is involved in NMO pathology. General significance Thus therapeutic strategies that target osteopontin signaling may be useful to treat NMO., Highlights • Osteopontin is significantly increased in the CSF of NMO patients. • Osteopontin is markedly elevated in the cerebral white matter of NMO patients. • NMO osteopontin is produced by astrocytes, neurons, oligodendroglia, and macrophages. • Interaction of NMO osteopontin with integrin αvβ3 promotes macrophage chemotaxis. • NMO osteopontin promotes macrophage chemotaxis by activating PI3K and MEK1/2.

We have investigated the regulation by divalent cations Mg 2+ , Ca 2+ and Mn 2+ of the functional activity of the human integrin VLA-1 expressed on neuroblastoma NB100 cells. VLA-1-mediated adhesion of NB100 cells to ligand collagen type I was supported by either mM concentrations of extracellular Mg 2+ or μM levels of Mn 2+ . In contrast, Ca 2+ alone did not induce activation of VLA-1 but exerted a potent inhibitory effect on the Mg 2+ -supported cell adhesion. We have also demonstrated that VLA-1 can be directly activated by the stimulatory monoclonal antibody TS2/16 specific for the integrin β1 subunit, resulting in effective adhesion of NB100 cells to type I collagen. This study has been possible by using a novel blocking VLA-α1 specific monoclonal antibody, 5E8D9.

Background Technological advances including high-throughput sequencing have identified numerous tumor-specific genetic changes in pediatric and adolescent cancers that can be exploited as targets for novel therapies. Scope of review This review provides a detailed overview of recent advances in the application of target-specific therapies for childhood cancers, either as single agents or in combination with other therapies. The review summarizes preclinical evidence on which clinical trials are based, early phase clinical trial results, and the incorporation of predictive biomarkers into clinical practice, according to cancer type. Major conclusions There is growing evidence that molecularly targeted therapies can valuably add to the arsenal available for treating childhood cancers, particularly when used in combination with other therapies. Nonetheless the introduction of molecularly targeted agents into practice remains challenging, due to the use of unselected populations in some clinical trials, inadequate methods to evaluate efficacy, and the need for improved preclinical models to both evaluate dosing and safety of combination therapies. General significance The increasing recognition of the heterogeneity of molecular causes of cancer favors the continued development of molecularly targeted agents, and their transfer to pediatric and adolescent populations., Highlights • Increasing numbers of targeted therapies are being tested for pediatric cancers. • Molecularly targeted therapies are proving most effective in combination regimes. • More rigorous preclinical testing should further improve clinical trial results.

The membrane bound metalloprotease aminopeptidase N (APN, CD13, EC 3.4.11.2) is a well established marker of normal and malignant cells of the myelo-monocytic lineage. It is also expressed by leukaemic blasts of a small group of patients suffering from acute or chronic lymphoid leukaemia. Recently, the expression of the APN gene in T cell lines as well as the induction of APN gene and surface expression in human peripheral T cells by mitogenic activation have been demonstrated. Here, by means of cytofluorimetric analysis evidence is provided, that the induction of APN surface expression is partially resistent to the action of the inhibitors of protein biosynthesis, puromycin and cycloheximide, and is not prevented by tunicamycin, an inhibitor of glycosylation. These data suggest that the rapid mitogen-induced surface expression of APN, detectable 20 hours after stimulation is dominated by mechanisms not dependent on de novo protein biosynthesis or glycosylation. As shown by simultaneous analyses, the inhibitors used did also differently modify the induction of surface expression of other inducible glycosylated leukocyte surface antigens, namely CD25, CD69 and CD95.

Background & Aims: Lipocalin 2 (Lcn2) is a multifunctional innate immune protein whose expression closely correlates with the extent of intestinal inflammation. However, whether Lcn2 plays a role in the pathogenesis of gut inflammation is unknown. Herein, we investigated the extent to which Lcn2 regulates inflammation and gut bacterial dysbiosis in mouse models of IBD. Methods: Lcn2 expression was monitored in murine colitis models and upon microbiota ablation/restoration. Wild-type (WT) and Lcn2 knockout (Lcn2KO) mice were analyzed for gut bacterial load, composition by 16S ribosomal RNA gene pyrosequencing, and their colitogenic potential by co-housing with interleukin (Il)10KO mice. Acute (dextran sodium sulfate) and chronic (IL10R neutralization and T-cell adoptive transfer) colitis were induced in WT and Lcn2KO mice with or without antibiotics. Results: Lcn2 expression was dramatically induced on inflammation and was dependent on the presence of a gut microbiota and MyD88 signaling. Use of bone marrowâchimeric mice showed that nonimmune cells are the major contributors of circulating Lcn2. Lcn2KO mice showed increased levels of entA-expressing gut bacteria burden, and, moreover, a broadly distinct bacterial community relative to WT littermates. Lcn2KO mice developed highly colitogenic T cells and showed exacerbated colitis on exposure to DSS or neutralization of IL10. Such exacerbated colitis could be prevented by antibiotic treatment. Moreover, exposure to the microbiota of Lcn2KO mice, via cohousing, resulted in severe colitis in Il10KO mice. Conclusions: Lcn2 is a bacterially induced, MyD88-dependent protein that plays an important role in gut homeostasis and a pivotal role on challenge. Hence, therapeutic manipulation of Lcn2 levels may provide a strategy to help manage diseases driven by alteration of the gut microbiota. Keywords: Gut Microbiota, Innate Immunity, Germ-Free Mice, MyD88, Colitis, NGAL, Siderocalin, Neutrophils, IBD