Studies on 6-chloro-5-(1-naphthyloxy)-2-(trifluoromethyl)-1H-benzimidazole/2-hydroxypropyl-β-cyclodextrin association: Characterization, molecular modeling studies, and in vivo anthelminthic activity

The purpose of this work is to study the molecular association that occurs between 2-hydroxypropyl-β-cyclodextrin (HPβCD) and 6-chloro-5-(1-naphthyloxy)-2-(trifluoromethyl)-1H-benzimidazole (RCB20), an antiparasitic compound recently found by our research group, with poor aqueous solubility. The complex stability constant and stoichiometric ratio determined by phase-solubility diagram and Job's plot provided evidence that HPβCD enhanced water solubility of RCB20 through inclusion complex formation. Two-dimensional 1H NMR spectroscopy is used to study the molecular arrangement of inclusion complex in solution. These results are further supported using molecular modeling studies. In the solid state, the complexation is confirmed by differential scanning calorimetry, powder X-ray diffraction, and scanning electron microscopy. Finally, RCB20/HPβCD complex has better activity than RCB20 against the adult and muscle larvae phase of Trichinella spiralis.     

Cell-cell contacts in the human cell line ECV304 exhibit both endothelial and epithelial characteristics

Endothelial cells separate the intra- and extravascular space and regulate transport processes between these compartments. Since intercellular junctions are required for these specific cell functions, the cell-cell contacts in the permanent cell line ECV304 were systematically analyzed and compared with human umbilical vein endothelial cells (HUVECs) in primary culture and with the epithelial Madin Darby Canine Kidney (MDCK) cell line. Filter-grown ECV304 cells generate a distinct electrical resistance and a permeability barrier between cell culture compartments. Electron microscopy of ECV304 cells revealed lateral membrane interdigitations, typically found in endothelial cells in vivo, with direct membrane contact sites, which prevented the diffusion of lanthanum. By immunoblot and immunofluorescence analysis, the expression and cellular localization of the tight junction and adherens-type junction proteins occludin, ZO-1, symplekin, beta-catenin, and plakoglobin were analyzed. ECV304 cells display further characteristics of endothelial cells, including the expresssion of thrombomodulin and of the vitronectin receptor CD51, as well as the secretion of plasminogen activator inhibitor 1 (PAI-1) and endothelin. However, ECV304 cells also express proteins characteristically found in epithelial cells, including E-cadherin and the desmosomal proteins desmoplakin, desmocollin, and desmoglein; occasionally desmosomal structures can be identified by electron microscopy. In conclusion, ECV304 cells express many endothelial markers and form specialized intercellular junctions that display some epithelial features. Thus this reportedly endothelial-derived permanent human cell line may be dedifferentiated toward an epithelial phenotype.     

Yersinia enterocolitica Infection and tcaA-Dependent Killing of Caenorhabditis elegans

Caenorhabditis elegans is a validated model to study bacterial pathogenicity. We report that Yersinia enterocolitica strains {"type":"entrez-nucleotide","attrs":{"text":"W22703","term_id":"1299536","term_text":"W22703"}}W22703 (biovar 2, serovar O:9) and WA314 (biovar 1B, serovar O:8) kill C. elegans when feeding on the pathogens for at least 15 min before transfer to the feeding strain Escherichia coli OP50. The killing by Yersinia enterocolitica requires viable bacteria and, in contrast to that by Yersinia pestis and Yersinia pseudotuberculosis strains, is biofilm independent. The deletion of tcaA encoding an insecticidal toxin resulted in an OP50-like life span of C. elegans, indicating an essential role of TcaA in the nematocidal activity of Y. enterocolitica. TcaA alone is not sufficient for nematocidal activity because E. coli DH5α overexpressing TcaA did not result in a reduced C. elegans life span. Spatial-temporal analysis of C. elegans infected with green fluorescent protein-labeled Y. enterocolitica strains showed that Y. enterocolitica colonizes the nematode intestine, leading to an extreme expansion of the intestinal lumen. By low-dose infection with {"type":"entrez-nucleotide","attrs":{"text":"W22703","term_id":"1299536","term_text":"W22703"}}W22703 or DH5α followed by transfer to E. coli OP50, proliferation of Y. enterocolitica, but not E. coli, in the intestinal lumen of the nematode was observed. The titer of {"type":"entrez-nucleotide","attrs":{"text":"W22703","term_id":"1299536","term_text":"W22703"}}W22703 cells within the worm increased to over 10⁶ per worm 4 days after infection while a significantly lower number of a tcaA knockout mutant was recovered. A strong expression of tcaA was observed during the first 5 days of infection. Y. enterocolitica WA314 (biovar 1B, serovar O:8) mutant strains lacking the yadA, inv, yopE, and irp1 genes known to be important for virulence in mammals were not attenuated or only slightly attenuated in their toxicity toward the nematode, suggesting that these factors do not play a significant role in the colonization and persistence of this pathogen in nematodes. In summary, this study supports the hypothesis that C. elegans is a natural host and nutrient source of Y. enterocolitica.Yersinia enterocolitica belongs to the family of Enterobacteriaceae and is a psychrotolerant human pathogen that causes gastrointestinal syndromes ranging from acute enteritis to mesenteric lymphadenitis (5). It infects a number of mammals, and swine was identified as a major source for human infection (6). A multiphasic life cycle, which comprises a free-living phase and several host-associated phases, including cold-blooded and warm-blooded hosts, appears to be characteristic for biovars 1B and 2 to 5 of Y. enterocolitica (7, 24).Nonmammalian host organisms including Dictyostelium discoideum, Drosophila melanogaster, or Caenorhabditis elegans are increasingly used to study host-pathogen interactions (16, 26). Due to the obvious parallels between the mammalian and invertebrate defense mechanisms, it has been suggested that the bacteria-invertebrate interaction has shaped the evolution of microbial pathogenicity (53). Several human pathogens including Gram-positive and Gram-negative bacteria infect and kill the soil nematode C. elegans when they are supplied as a nutrient source (42). For example, Streptococcus pneumoniae (4), Listeria monocytogenes (50), extraintestinal Escherichia coli (15), and Staphylococcus aureus (43) but not Bacillus subtilis have been shown to kill the nematode. Upon infection of C. elegans with Enterococcus faecalis, Gram-positive virulence-related factors as well as putative antimicrobials have been identified (20, 35). The extensive conservation in virulence mechanisms directed against invertebrates as well as mammals was demonstrated using a screen with Pseudomonas aeruginosa (30). In this study, 10 of 13 genes whose knockout attenuated the nematode killing were also required for full virulence in a mouse model, confirming the suitability of the C. elegans model to study bacterial pathogenicity. C. elegans is also colonized by Salmonella enterica serovar Typhimurium (S. Typhimurium). This process requires Salmonella virulence factors and was used to study the innate immune response of the nematode (1, 2, 49).The effect of pathogenic Yersinia spp. on C. elegans has also been investigated. It could be demonstrated that both Yersinia pestis and Yersinia pseudotuberculosis block food intake by creating a biofilm around the worm''s mouth (13, 27). This biofilm formation requires the hemin storage locus (hms) and has been suggested to be responsible for the blockage of the digestive tract following uptake by fleas, thus acting as a bacterial defense against predation by invertebrates. In a study with 40 Y. pseudotuberculosis strains, one-quarter of them caused an infection of C. elegans by biofilm formation on the worm head (27). In contrast, a similar effect was not observed following nematode infection with 15 Y. enterocolitica strains. Using a Y. pestis strain lacking the hms genes, it could be demonstrated that this mutant can infect and kill the nematode by a biofilm-independent mechanism that includes the accumulation of Y. pestis in the intestine of the worm (47). This pathogenesis model was applied to show that putative virulence factors such as YapH, OmpT, or a metalloprotease, Y3857, but not the virulence plasmids pCD1 and pPCP1, are required for Y. pestis virulence in C. elegans. Six yet unknown genes required for full virulence in C. elegans were also identified, and one of them appeared to be a virulence factor in the mouse infection model.C. elegans has not been used to study the pathogenicity properties of Y. enterocolitica, mainly due to the fact that many of its virulence factors are upregulated at 37°C in comparison to growth at lower temperatures while C. elegans cannot be cultivated at temperatures above 25°C. In this study, we examined for the first time the infection of C. elegans by Y. enterocolitica strains, demonstrating that this pathogen colonizes and kills C. elegans and that the insecticidal toxin TcaA, which is expressed only at ambient temperature, is required for full nematocidal activity.     

Improved monovalent TNF receptor 1-selective inhibitor with novel heterodimerizing Fc

The development of alternative therapeutic strategies to tumor necrosis factor (TNF)-blocking antibodies for the treatment of inflammatory diseases has generated increasing interest. In particular, selective inhibition of TNF receptor 1 (TNFR1) promises a more precise intervention, tackling only the pro-inflammatory responses mediated by TNF while leaving regenerative and pro-survival signals transduced by TNFR2 untouched. We recently generated a monovalent anti-TNFR1 antibody fragment (Fab 13.7) as an efficient inhibitor of TNFR1. To improve the pharmacokinetic properties of Fab 13.7, the variable domains of the heavy and light chains were fused to the N-termini of newly generated heterodimerizing Fc chains. This novel Fc heterodimerization technology, designated “Fc-one/kappa” (Fc1κ) is based on interspersed constant Ig domains substituting the CH3 domains of a γ1 Fc. The interspersed immunoglobulin (Ig) domains originate from the per se heterodimerizing constant CH1 and CLκ domains and contain sequence stretches of an IgG1 CH3 domain, destined to enable interaction with the neonatal Fc receptor, and thus promote extended serum half-life. The resulting monovalent Fv-Fc1κ fusion protein (Atrosimab) retained strong binding to TNFR1 as determined by enzyme-linked immunosorbent assay and quartz crystal microbalance, and potently inhibited TNF-induced activation of TNFR1. Atrosimab lacks agonistic activity for TNFR1 on its own and in the presence of anti-human IgG antibodies and displays clearly improved pharmacokinetic properties.     

Identification and validation of colorectal neoplasia-specific methylation markers for accurate classification of disease

Aberrant DNA methylation occurs early in oncogenesis, is stable, and can be assayed in tissues and body fluids. Therefore, genes with aberrant methylation can provide clues for understanding tumor pathways and are attractive candidates for detection of early neoplastic events. Identification of sequences that optimally discriminate cancer from other diseased and healthy tissues is needed to advance both approaches. Using well-characterized specimens, genome-wide methylation techniques were used to identify candidate markers specific for colorectal neoplasia. To further validate 30 of these candidates from genome-wide analysis and 13 literature-derived genes, including genes involved in cancer and others with unknown functions, a high-throughput methylation-specific oligonucleotide microarray was used. The arrays were probed with bisulfite-converted DNA from 89 colorectal adenocarcinomas, 55 colorectal polyps, 31 inflammatory bowel disease, 115 extracolonic cancers, and 67 healthy tissues. The 20 most discriminating markers were highly methylated in colorectal neoplasia (area under the receiver operating characteristic curve > 0.8; P < 0.0001). Normal epithelium and extracolonic cancers revealed significantly lower methylation. Real-time PCR assays developed for 11 markers were tested on an independent set of 149 samples from colorectal adenocarcinomas, other diseases, and healthy tissues. Microarray results could be reproduced for 10 of 11 marker assays, including eight of the most discriminating markers (area under the receiver operating characteristic curve > 0.72; P < 0.009). The markers with high specificity for colorectal cancer have potential as blood-based screening markers whereas markers that are specific for multiple cancers could potentially be used as prognostic indicators, as biomarkers for therapeutic response monitoring or other diagnostic applications, compelling further investigation into their use in clinical testing and overall roles in tumorigenesis.     

Metabolic profiling of rhizomes of native populations of the strictly endemic Croatian species Iris adriatica

Iris adriatica Trinajsti? ex Miti? (Iridaceae L.) is a strictly endemic taxon from Croatia. It is a rhizomatous dwarf plant from the I. pumila complex with a distribution area limited to the Croatian part of the Mediterranean area, mainly central Dalmatia. The genus Iris is known for its richness in isoflavonoids which also play a significant role in chemotaxonomy and biological activity. Hence, in the current study, different plant batches of I. adriatica collected in early spring of 2016 were analysed for their phytochemical profiles and qualitatively compared. UHPLC-PDA-ESI-MS analyses of methanolic rhizome extracts were performed. Altogether, 36 compounds, representing isoflavonoids (including 6,7-methylendioxy derivatives), benzophenones and xanthones were found as aglycones or in glycosidically bound form to be the main constituent groups of I. adriatica rhizomes. Qualitative results were identical between different batches of plant material from collection sites in central Dalmatia, they differed only in quantity. For some phenolic compounds of I. adriatica, chemotaxonomic relevance was detected.     

Restricted aeroallergen access to airway mucosal dendritic cells in vivo limits allergen-specific CD4+ T cell proliferation during the induction of inhalation tolerance

Chronic innocuous aeroallergen exposure attenuates CD4(+) T cell-mediated airways hyperresponsiveness in mice; however, the mechanism(s) remain unclear. We examined the role of airway mucosal dendritic cell (AMDC) subsets in this process using a multi-OVA aerosol-induced tolerance model in sensitized BALB/c mice. Aeroallergen capture by both CD11b(lo) and CD11b(hi) AMDC and the delivery of OVA to airway draining lymph nodes by CD8α(-) migratory dendritic cells (DC) were decreased in vivo (but not in vitro) when compared with sensitized but nontolerant mice. This was functionally significant, because in vivo proliferation of OVA-specific CD4(+) T cells was suppressed in airway draining lymph nodes of tolerized mice and could be restored by intranasal transfer of OVA-pulsed and activated exogenous DC, indicating a deficiency in Ag presentation by endogenous DC arriving from the airway mucosa. Bone marrow-derived DC Ag-presenting function was suppressed in multi-OVA tolerized mice, and allergen availability to airway APC populations was limited after multi-OVA exposure, as indicated by reduced OVA and dextran uptake by airway interstitial macrophages, with diffusion rather than localization of OVA across the airway mucosal surface. These data indicate that inhalation tolerance limits aeroallergen capture by AMDC subsets through a mechanism of bone marrow suppression of DC precursor function coupled with reduced Ag availability in vivo at the airway mucosa, resulting in limited Ag delivery to lymph nodes and hypoproliferation of allergen-specific CD4(+) T cells.     

Adaptation of the Ras-recruitment system to the analysis of interactions between membrane-associated proteins

Interactions of membrane-associated proteins play important roles in many cellular processes. The yeast two-hybrid assay is of limited utility for the analysis of such interactions, due to the need for soluble protein partners, whose interaction is assessed in the nucleus. The advent of the Ras-recruitment system (RRS) has enabled the study of membrane-associated proteins interacting with cytoplasmic proteins fused to Ras. Constitutive membrane association of the Ras fusion protein is expected to complement the growth defect of the yeast strain CDC25-2, assayed in the RRS, independent from the interaction with a membrane-bound partner. We describe the adaptation of the RRS to the analysis of interactions between two membrane-associated proteins using a model system. These results may facilitate the study of protein–protein interactions between membrane-bound proteins and further increase the utility of the RRS.