T cell interaction with ICAM-1-deficient endothelium in vitro: essential role for ICAM-1 and ICAM-2 in transendothelial migration of T cells

Transendothelial migration is a crucial step in the complex process of lymphocyte extravasation during lymphocyte homing, immunosurveillance and inflammation. However, little is known about the precise role of cell adhesion molecules (CAM) involved in this particular event. To define the CAM involved in T cell adhesion versus transendothelial migration, we have previously established an in vitro transendothelial migration system using mouse T cells and mouse endothelioma cells. We demonstrate here that, using ICAM-1-deficient endothelioma cells derived from ICAM-1 mutant mice, transendothelial migration of T cells was inhibited to a much greater extent when compared to migration across wild-type cells treated with a blocking anti-ICAM-1 monoclonal antibody. This unexpected result was confirmed by a rescue experiment using retroviral transfer of wild-type ICAM-1 into ICAM-1-deficient endothelial cells. Additional experiments showed that, in the absence of functional ICAM-1, only ICAM-2 was involved in transendothelial migration, but not PECAM-1, VCAM-1, or E-selectin. Taking this novel approach, we show that ICAM-1 and ICAM-2 are essential for transendothelial migration of T cells.

Solution structure and interaction of cupiennin 1a, a spider venom peptide, with phospholipid bilayers

The solution structure of cupiennin 1a, a 35 residue, basic antibacterial peptide isolated from the venom of the spider Cupiennius salei, has been determined by nuclear magnetic resonance (NMR) spectroscopy. The peptide was found to adopt a helix−hinge−helix structure in a membrane mimicking solvent. The hinge may play a role in allowing the amphipathic N-terminal helix and polar C-terminal helix to orient independently upon membrane binding, in order to achieve maximal antibacterial efficacy. Solid-state 31P and 2H NMR was used to further study the effects of cupiennin 1a on the dynamic properties of lipid membranes, using zwitterionic chain deuterated dimyristoylphosphatidylcholine (d54-DMPC) and anionic dimyristoylphosphatidylglycerol (DMPG) multilamellar vesicles. In d54-DMPC alone, cupiennin 1a caused a decrease in the 31P chemical shift anisotropy, indicating some interaction with the lipid head groups, and a decrease in order over the entire acyl chain. In contrast, for the mixed (d54-DMPC/DMPG) lipid system cupiennin 1a appeared to induce lateral separation of the two lipids as evidenced by the 31P spectra, in which the peptide preferentially interacted with DMPG. Little effect was observed on the deuterated acyl chain order parameters in the d54-DMPC/DMPG model membranes. Furthermore, 31P NMR relaxation measurements confirmed a differential effect on the lipid motions depending upon the membrane composition. Therefore, subtle differences are likely in the mechanism by which cupiennin 1a causes membrane lysis in either prokaryotic or eukaryotic cells, and may explain the specific spectrum of activity.

Topical caspofungin for treatment of keratitis caused by Candida albicans in a rabbit model

Candida albicans is the most frequent cause of fungal keratitis in temperate regions. Caspofungin has potent activity against Candida spp. in a variety of clinical settings. Little is known, however, about its activity against fungal keratitis. We compared the efficacy of topical caspofungin with that of topical amphotericin B (AMB) in a rabbit model of experimental keratomycosis. Keratitis was induced with a standardized inoculum of Candida albicans (SC 5314) placed on the debrided cornea. Twenty-four hours after infection, animals were randomly assigned to treatment with 0.15% caspofungin, 0.5% caspofungin, 0.15% AMB, and a saline control (n = 12 rabbits in each group). For the first 12 h, treatment was repeated every 30 min and, after a 12-h pause, was resumed at hourly intervals for another 12 h. The animals were examined and killed 12 h after administration of the last dose. Treatment effects were evaluated by clinical assessment, fungal culture, and histopathology. Drug treatment significantly reduced corneal fungal recovery from 3.78 log10 CFU in saline-treated animals to 2.97, 1.76, and 1.18 log10 CFU in animals treated with 0.15% caspofungin, 0.5% caspofungin, and 0.15% AMB, respectively. By histopathology, the mean hyphal density was significantly lower in the corneas of treated animals than in those of the controls; there was no difference in hyphal densities between the different treatment groups. The depth of corneal invasion was not significantly reduced by the antifungal treatments. By clinical assessment, keratitis progressed in animals treated with saline, whereas disease progression was inhibited by all drug treatment regimens. In our rabbit model, 0.5% caspofungin was as effective as 0.15% AMB for the topical treatment of Candida keratitis. The potential clinical efficacy of caspofungin awaits further investigation.

Candida species distribution and antifungal susceptibility testing according to European Committee on Antimicrobial Susceptibility Testing and new vs. old Clinical and Laboratory Standards Institute clinical breakpoints: a 6-year prospective candidaemia survey from the fungal infection network of Switzerland

We analyzed the species distribution of Candida blood isolates (CBIs), prospectively collected between 2004 and 2009 within FUNGINOS, and compared their antifungal susceptibility according to clinical breakpoints defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) in 2013, and the Clinical and Laboratory Standards Institute (CLSI) in 2008 (old CLSI breakpoints) and 2012 (new CLSI breakpoints). CBIs were tested for susceptiblity to fluconazole, voriconazole and caspofungin by microtitre broth dilution (Sensititre® YeastOne™ test panel). Of 1090 CBIs, 675 (61.9%) were C. albicans, 191 (17.5%) C. glabrata, 64 (5.9%) C. tropicalis, 59 (5.4%) C. parapsilosis, 33 (3%) C. dubliniensis, 22 (2%) C. krusei and 46 (4.2%) rare Candida species. Independently of the breakpoints applied, C. albicans was almost uniformly (>98%) susceptible to all three antifungal agents. In contrast, the proportions of fluconazole- and voriconazole-susceptible C. tropicalis and F-susceptible C. parapsilosis were lower according to EUCAST/new CLSI breakpoints than to the old CLSI breakpoints. For caspofungin, non-susceptibility occurred mainly in C. krusei (63.3%) and C. glabrata (9.4%). Nine isolates (five C. tropicalis, three C. albicans and one C. parapsilosis) were cross-resistant to azoles according to EUCAST breakpoints, compared with three isolates (two C. albicans and one C. tropicalis) according to new and two (2 C. albicans) according to old CLSI breakpoints. Four species (C. albicans, C. glabrata, C. tropicalis and C. parapsilosis) represented >90% of all CBIs. In vitro resistance to fluconazole, voriconazole and caspofungin was rare among C. albicans, but an increase of non-susceptibile isolates was observed among C. tropicalis/C. parapsilosis for the azoles and C. glabrata/C. krusei for caspofungin according to EUCAST and new CLSI breakpoints compared with old CLSI breakpoints.

Phosphorylation of serine 4642 in the C-terminus of plectin by MNK2 and PKA modulates its interaction with intermediate filaments

Plectin is a versatile cytolinker of the plakin family conferring cell resilience to mechanical stress in stratified epithelia and muscles. It acts as a critical organizer of the cytoskeletal system by tethering various intermediate filament (IF) networks through its C-terminal IF-binding domain (IFBD). Mutations affecting the IFBD cause devastating human diseases. Here, we show that serine 4642, which is located in the extreme C-terminus of plectin, is phosphorylated in different cell lines. Phosphorylation of S4642 decreased the ability of plectin IFBD to associate with various IFs, as assessed by immunofluorescence microscopy and cell fractionation studies, as well as in yeast two-hybrid assays. Plectin phosphorylated at S4642 was reduced at sites of IF network anchorage along cell-substrate contacts in both skin and cultured keratinocytes. Treatment of SK-MEL-2 and HeLa cells with okadaic acid increased plectin S4642 phosphorylation, suggesting that protein phosphatase 2A dephosphorylates this residue. Moreover, plectin S4642 phosphorylation was enhanced after cell treatment with EGF, phorbol ester, sorbitol and 8-bromo-cyclic AMP, as well as during wound healing and protease-mediated cell detachment. Using selective protein kinase inhibitors, we identified two different kinases that modulate the phosphorylation of plectin S4642 in HeLa cells: MNK2, which is downstream of the ERK1/2-dependent MAPK cascade, and PKA. Our study indicates that phosphorylation of S4642 has an important regulatory role in the interaction of plectin with IFs and identifies a novel link between MNK2 and the cytoskeleton.

GATA-4 and GATA-6 modulate tissue-specific transcription of the human gene for P450c17 by direct interaction with Sp1.

Cytochrome P450c17 catalyzes steroidogenic 17alpha-hydroxylase and 17,20 lyase activities. Expression of the gene for P450c17 is cAMP dependent, tissue specific, developmentally programmed, and varies among species. Binding of Sp1, Sp3, and NF1-C (nuclear factor 1-C) to the first 227 bp of 5'flanking DNA (-227/LUC) is crucial for basal transcription in human NCI-H295A adrenal cells. Human placental JEG-3 cells contain Sp1, Sp3, and NF1, but do not express -227/LUC, even when transfected with a vector expressing steroidogenic factor 1 (SF-1). Therefore, other factors are essential for basal expression of P450c17. Deoxyribonuclease I footprinting and EMSAs identified a GATA consensus site at -64/-58 and an SF-1 site at -58/-50. RT-PCR identified GATA-4, GATA-6, and SF-1 in NCI-H295A cells and GATA-2 and GATA-3, but not GATA-4, GATA-6, or SF-1 in JEG-3 cells. Cotransfection of either GATA-4 or GATA-6 without SF-1 activated -227/LUC in JEG-3 cells, but cotransfection of GATA-2 or GATA-3 with or without SF-1 did not. Surprisingly, mutation of the GATA binding site in -227/LUC increased GATA-4 or GATA-6 induced activity, whereas mutation of the Sp1/Sp3 site decreased it. Furthermore, promoter constructs including the GATA site, but excluding the Sp1/Sp3 site at -196/-188, were not activated by GATA-4 or GATA-6, suggesting an interaction between Sp1/Sp3 and GATA-4 or GATA-6. Glutathione-S-transferase pull-down experiments and coimmunoprecipitation demonstrated interaction between GATA-4 or GATA-6 and Sp1, but not Sp3. Chromatin immunoprecipitation assays confirmed that this GATA-4/6 interaction with Sp1 occurred at the Sp site in the P450c17 promoter in NCI-H295A cells. Demethylation with 5-aza-2-deoxycytidine permitted JEG-3 cells to express endogenous P450c17, SF-1, GATA-4, GATA-6, and transfected -227/LUC. Thus, GATA-4 or GATA-6 and Sp1 together regulate expression of P450c17 in adrenal NCI-H295A cells and methylation of P450c17, GATA-4 and GATA-6 silence the expression of P450c17 in placental JEG-3 cells.

Three-dimensional imaging methods for quantitative analysis of facial soft tissues and skeletal morphology in patients with orofacial clefts: a systematic review.

BACKGROUND Current guidelines for evaluating cleft palate treatments are mostly based on two-dimensional (2D) evaluation, but three-dimensional (3D) imaging methods to assess treatment outcome are steadily rising. OBJECTIVE To identify 3D imaging methods for quantitative assessment of soft tissue and skeletal morphology in patients with cleft lip and palate. DATA SOURCES Literature was searched using PubMed (1948-2012), EMBASE (1980-2012), Scopus (2004-2012), Web of Science (1945-2012), and the Cochrane Library. The last search was performed September 30, 2012. Reference lists were hand searched for potentially eligible studies. There was no language restriction. STUDY SELECTION We included publications using 3D imaging techniques to assess facial soft tissue or skeletal morphology in patients older than 5 years with a cleft lip with/or without cleft palate. We reviewed studies involving the facial region when at least 10 subjects in the sample size had at least one cleft type. Only primary publications were included. DATA EXTRACTION Independent extraction of data and quality assessments were performed by two observers. RESULTS Five hundred full text publications were retrieved, 144 met the inclusion criteria, with 63 high quality studies. There were differences in study designs, topics studied, patient characteristics, and success measurements; therefore, only a systematic review could be conducted. Main 3D-techniques that are used in cleft lip and palate patients are CT, CBCT, MRI, stereophotogrammetry, and laser surface scanning. These techniques are mainly used for soft tissue analysis, evaluation of bone grafting, and changes in the craniofacial skeleton. Digital dental casts are used to evaluate treatment and changes over time. CONCLUSION Available evidence implies that 3D imaging methods can be used for documentation of CLP patients. No data are available yet showing that 3D methods are more informative than conventional 2D methods. Further research is warranted to elucidate it.

FUS/TLS contributes to replication-dependent histone gene expression by interaction with U7 snRNPs and histone-specific transcription factors.

Replication-dependent histone genes are up-regulated during the G1/S phase transition to meet the requirement for histones to package the newly synthesized DNA. In mammalian cells, this increment is achieved by enhanced transcription and 3' end processing. The non-polyadenylated histone mRNA 3' ends are generated by a unique mechanism involving the U7 small ribonucleoprotein (U7 snRNP). By using affinity purification methods to enrich U7 snRNA, we identified FUS/TLS as a novel U7 snRNP interacting protein. Both U7 snRNA and histone transcripts can be precipitated by FUS antibodies predominantly in the S phase of the cell cycle. Moreover, FUS depletion leads to decreased levels of correctly processed histone mRNAs and increased levels of extended transcripts. Interestingly, FUS antibodies also co-immunoprecipitate histone transcriptional activator NPAT and transcriptional repressor hnRNP UL1 in different phases of the cell cycle. We further show that FUS binds to histone genes in S phase, promotes the recruitment of RNA polymerase II and is important for the activity of histone gene promoters. Thus, FUS may serve as a linking factor that positively regulates histone gene transcription and 3' end processing by interacting with the U7 snRNP and other factors involved in replication-dependent histone gene expression.

Multivalency effects in Pseudomonas aeruginosa biofilm inhibition and dispersal by glycopeptide dendrimers targeting lectin LecA

The galactose specific lectin LecA partly mediates the formation of antibiotic resistant biofilms by Pseudomonas aeruginosa, an opportunistic pathogen causing lethal airways infections in immunocompromised and cystic fibrosis patients, suggesting that preventing LecA binding to natural saccharides might provide new opportunities for treatment. Here 8-fold (G3) and 16-fold (G4) galactosylated analogs of GalAG2, a tetravalent G2 glycopeptide dendrimer LecA ligand and P. aeruginosa biofilm inhibitor, were obtained by convergent chloroacetyl thioether (ClAc) ligation between 4-fold or 8-fold chloroacetylated dendrimer cores and digalactosylated dendritic arms. Hemagglutination inhibition, isothermal titration calorimetry and biofilm inhibition assays showed that G3 dendrimers bind LecA slightly better than their parent G2 dendrimers and induce complete biofilm inhibition and dispersal of P. aeruginosa biofilms, while G4 dendrimers show reduced binding and no biofilm inhibition. A binding model accounting for the observed saturation of glycopeptide dendrimer galactosyl groups and LecA binding sites is proposed based on the crystal structure of a G3 dendrimer LecA complex.

Invited review: Growth-promoting effects of colostrum in calves based on interaction with intestinal cell surface receptors and receptor-like transporters

The postnatal development and maturation of the gastrointestinal (GI) tract of neonatal calves is crucial for their survival. Major morphological and functional changes in the calf's GI tract initiated by colostrum bioactive substances promote the establishment of intestinal digestion and absorption of food. It is generally accepted that colostrum intake provokes the maturation of organs and systems in young calves, illustrating the significance of the cow-to-calf connection at birth. These postnatal adaptive changes of the GI tissues in neonatal calves are especially induced by the action of bioactive substances such as insulin-like growth factors, hormones, or cholesterol carriers abundantly present in colostrum. These substances interact with specific cell-surface receptors or receptor-like transporters expressed in the GI wall of neonatal calves to elicit their biological effects. Therefore, the abundance and activity of cell surface receptors and receptor-like transporters binding colostral bioactive substances are a key aspect determining the effects of the cow-to-calf connection at birth. The present review compiles the information describing the effects of colostrum feeding on selected serum metabolic and endocrine traits in neonatal calves. In this context, the current paper discusses specifically the consequences of colostrum feeding on the GI expression and activity of cell-receptors and receptor-like transporters binding growth hormone, insulin-like growth factors, insulin, or cholesterol acceptors in neonatal calves.

Morphology and dynamics of the jets of comet 67P/Churyumov-Gerasimenko: Early-phase development

Aims. The OSIRIS camera onboard the Rosetta spacecraft obtained close-up views of the dust coma of comet 67P. The jet structures can be used to trace their source regions and to examine the possible effect of gas-surface interaction. Methods. We analyzed the wide-angle images obtained in the special dust observation sequences between August and September 2014. The jet features detected in different images were compared to study their time variability. The locations of the potential source regions of some of the jets are identified by ray tracing. We used a ring-masking technique to calculate the brightness distribution of dust jets along the projected distance. Results. The jets detected between August and September 2014 mostly originated in the Hapi region. Morphological changes appeared over a timescale of several days in September. The brightness slope of the dust jets is much steeper than the background coma. This might be related to the sublimation or fragmentation of the emitted dust grains. Interaction of the expanding gas flow with the cliff walls on both sides of Hapi could lead to erosion and material down-fall to the nucleus surface.