Production of interferon-gamma by activated T-cell receptor-alphabeta CD8alphabeta intestinal intraepithelial lymphocytes is required and sufficient for disruption of the intestinal barrier integrity

Maintenance of intestinal epithelial barrier function is of vital importance in preventing uncontrolled influx of antigens and the potentially ensuing inflammatory disorders. Intestinal intraepithelial lymphocytes (IEL) are in intimate contact with epithelial cells and may critically regulate the epithelial barrier integrity. While a preserving impact has been ascribed to the T-cell receptor (TCR)-gammadelta subset of IEL, IEL have also been shown to attenuate the barrier function. The present study sought to clarify the effects of IEL by specifically investigating the influence of the TCR-alphabeta CD8alphabeta and TCR-alphabeta CD8alphaalpha subsets of IEL on the intestinal epithelial barrier integrity. To this end, an in vitro coculture system of the murine intestinal crypt-derived cell-line mIC(cl2) and syngeneic ex vivo isolated IEL was employed. Epithelial integrity was assessed by analysis of transepithelial resistance (TER) and paracellular flux of fluorescein isothiocyanate-conjugated (FITC-) dextran. The TCR-alphabeta CD8alphaalpha IEL and resting TCR-alphabeta CD8alphabeta IEL did not affect TER of mIC(cl2) or flux of FITC-dextran. In contrast, activated TCR-alphabeta CD8alphabeta IEL clearly disrupted the integrity of the mIC(cl2) monolayer. No disrupting effect was seen with activated TCR-alphabeta CD8alphabeta IEL from interferon-gamma knockout mice. These findings demonstrate that secretion of interferon-gamma by activated TCR-alphabeta CD8alphabeta IEL is strictly required and also sufficient for disrupting the intestinal epithelial barrier function.

Bone healing around titanium implants in two rat colitis models

OBJECTIVE Crohn's disease is a chronic inflammatory process that has recently been associated with a higher risk of early implant failure. Herein we provide information on the impact of colitis on peri-implant bone formation using preclinical models of chemically induced colitis. METHODS Colitis was induced by intrarectal instillation of 2,4,6-trinitro-benzene-sulfonic-acid (TNBS). Colitis was also induced by feeding rats dextran-sodium-sulfate (DSS) in drinking water. One week after disease induction, titanium miniscrews were inserted into the tibia. Four weeks after implantation, peri-implant bone volume per tissue volume (BV/TV) and bone-to-implant contacts (BIC) were determined by histomorphometric analysis. RESULTS Cortical histomorphometric parameters were similar in the control (n = 10), DSS (n = 10) and TNBS (n = 8) groups. Cortical BV/TV was 92.2 ± 3.7%, 92.0 ± 3.0% and 92.6 ± 2.7%. Cortical BIC was 81.3 ± 8.8%, 83.2 ± 8.4% and 84.0 ± 7.0%, respectively. No significant differences were observed when comparing the medullary BV/TV and BIC (19.5 ± 6.4%, 16.2 ± 5.6% and 15.4 ± 9.0%) and (48.8 ± 12.9%, 49.2 ± 6.2 and 41.9 ± 11.7%), respectively. Successful induction of colitis was confirmed by loss of body weight and colon morphology. CONCLUSIONS The results suggest bone regeneration around implants is not impaired in chemically induced colitis models. Considering that Crohn's disease can affect any part of the gastrointestinal tract including the mouth, our model only partially reflects the clinical situation.

The substrate degradome of meprin metalloproteases reveals an unexpected proteolytic link between meprin β and ADAM10.

The in vivo roles of meprin metalloproteases in pathophysiological conditions remain elusive. Substrates define protease roles. Therefore, to identify natural substrates for human meprin α and β we employed TAILS (terminal amine isotopic labeling of substrates), a proteomics approach that enriches for N-terminal peptides of proteins and cleavage fragments. Of the 151 new extracellular substrates we identified, it was notable that ADAM10 (a disintegrin and metalloprotease domain-containing protein 10)-the constitutive α-secretase-is activated by meprin β through cleavage of the propeptide. To validate this cleavage event, we expressed recombinant proADAM10 and after preincubation with meprin β, this resulted in significantly elevated ADAM10 activity. Cellular expression in murine primary fibroblasts confirmed activation. Other novel substrates including extracellular matrix proteins, growth factors and inhibitors were validated by western analyses and enzyme activity assays with Edman sequencing confirming the exact cleavage sites identified by TAILS. Cleavages in vivo were confirmed by comparing wild-type and meprin(-/-) mice. Our finding of cystatin C, elafin and fetuin-A as substrates and natural inhibitors for meprins reveal new mechanisms in the regulation of protease activity important for understanding pathophysiological processes.

TREM-1 deficiency can attenuate disease severity without affecting pathogen clearance

Triggering receptor expressed on myeloid cells-1 (TREM-1) is a potent amplifier of pro-inflammatory innate immune reactions. While TREM-1-amplified responses likely aid an improved detection and elimination of pathogens, excessive production of cytokines and oxygen radicals can also severely harm the host. Studies addressing the pathogenic role of TREM-1 during endotoxin-induced shock or microbial sepsis have so far mostly relied on the administration of TREM-1 fusion proteins or peptides representing part of the extracellular domain of TREM-1. However, binding of these agents to the yet unidentified TREM-1 ligand could also impact signaling through alternative receptors. More importantly, controversial results have been obtained regarding the requirement of TREM-1 for microbial control. To unambiguously investigate the role of TREM-1 in homeostasis and disease, we have generated mice deficient in Trem1. Trem1(-/-) mice are viable, fertile and show no altered hematopoietic compartment. In CD4(+) T cell- and dextran sodium sulfate-induced models of colitis, Trem1(-/-) mice displayed significantly attenuated disease that was associated with reduced inflammatory infiltrates and diminished expression of pro-inflammatory cytokines. Trem1(-/-) mice also exhibited reduced neutrophilic infiltration and decreased lesion size upon infection with Leishmania major. Furthermore, reduced morbidity was observed for influenza virus-infected Trem1(-/-) mice. Importantly, while immune-associated pathologies were significantly reduced, Trem1(-/-) mice were equally capable of controlling infections with L. major, influenza virus, but also Legionella pneumophila as Trem1(+/+) controls. Our results not only demonstrate an unanticipated pathogenic impact of TREM-1 during a viral and parasitic infection, but also indicate that therapeutic blocking of TREM-1 in distinct inflammatory disorders holds considerable promise by blunting excessive inflammation while preserving the capacity for microbial control.

Lectin binding patterns in two cultured endothelial cell types derived from bovine corpus luteum

Epithelial cells of different phenotypes derived from bovine corpus luteum have been studied intensively in our laboratory. In this study, specific lectin binding was examined for cells of type 1 and 3, which were defined as endothelial cells. In order to confirm differences in their glycocalyx at the light microscopic level, five biotinylated lectins were applied to postconfluent cultures which had been fixed with buffered paraformaldehyde or glutaraldehyde. Cells were not permeabilized with any detergent. Lectin binding was localized with a streptavidin-peroxidase complex which was visualized with two different techniques. The DAB technique detected peroxidase histochemically, while the immunogold technique used an anti-peroxidase gold complex together with silver amplification. Neither cell type 1 nor cell type 3 bound a particular lectin selectively, yet each cell type expressed a particular lectin binding pattern. With the DAB technique, diverse lectin binding patterns were seen, probably indicating either "outside" binding, i.e., a diffuse pattern, a lateral-cell-side pattern and a microvillus-like pattern, or "inside" binding, i.e., a diffuse pattern, and a granule-like pattern. With the immunogold technique, only "outside" binding was observed. In addition, the patterns of single cilia or of single circles were detected, the latter roughly representing 3-micron-sized binding sites for concanavalin A. When localizing them at the ultrastructural level, single circles corresponded with micron-sized discontinuities of the plasma membrane. Shedding vesicles were detected whose outer membrane was labelled with concanavalin A. Our results confirm the diversity of the two cell types under study. The "inside" lectin binding may be caused by way of transient plasma membrane openings and related to shedding of right-side out vesicles ("ectocytosis").

Phagocytic uptake of Encephalitozoon cuniculi by nonprofessional phagocytes

Encephalitozoon cuniculi is an obligate intracellular, spore-forming parasite belonging to the microsporidia that can cause disseminated infection in immunocompromised persons. E. cuniculi spores infect host cells by germination, i.e., by explosively everting the polar filament, through which the spore contents (sporoplasms) are subsequently injected into the cytoplasm. In addition, we observed intracellular, nongerminated spores in various nonprofessional phagocytes. In MRC5 cells, the number of internalized spores was approximately 10-fold higher than the number of injected sporoplasms. Compared to the rate of uptake by human monocyte-derived macrophages, internalization rates by A549 cells, MRC5 cells, and 293 cells were 0.6, 4.4, and 22.2%, respectively. The mechanism of uptake was studied in MRC5 cells. Killed spores were internalized at the same rate as live spores, indicating that nongerminated parasites do not actively participate in cell entry. Cytochalasin D inhibited uptake of spores by 95%, demonstrating an actin-dependent process. By electron and epifluorescence microscopy, intracellular spores were found in a tightly fitting membrane-bound compartment. The vacuole containing the spores was positive for the lysosomal membrane protein LAMP-1 and colocalized with the late endosomal-lysosomal content marker rhodamine dextran. Our results show that, in addition to the unique way in which microsporidia infect cells, E. cuniculi spores enter nonprofessional phagocytes by phagocytosis and traffic into a late endosomal-lysosomal compartment.

Development of light-responsive porous polycarbonate membranes for controlled caffeine delivery

For controlled caffeine release, light-responsive membranes were developed. It was possible to produce membranes that reduced their caffeine permeability resistance by about 97% when irradiated with UV-light compared to measurements at daylight. This was achieved by grafting polymers possessing photochromic units onto track-edged polycarbonate membranes. Covalently linked coatings on porous polycarbonate membranes were obtained by plasma activation of the membrane surface followed by plasma-induced graft polymerization. Copolymerization of spiro-compounds during the coating process as well as postmodification of preformed coatings with spiropyran resulted in photochromic membranes. For the copolymerization process, the synthesis of five photochromic methacrylic and acrylic spiropyrans and spirooxazines was successfully performed. Additionally, a spiropyran with carboxylic acid functionality was synthesized for the postmodification process. This enabled us to postmodify polymeric materials containing alcohol or amine groups to obtain photochromic materials. UV-irradiation of these light-responsive membranes resulted in a strong colouration of the membrane, in a reduction of surface tension, which resulted in a decreased caffeine permeability resistance. The membranes were characterized using XPS for the elemental composition of the coating, contact angle measurements for the surface tension, solid-state UV/VIS measurements for the determination of the kinetic and stability properties, and two-photon microscopy for the localisation of the photochromic substance in the porous membrane.

The Early Activation Marker CD69 Regulates the Expression of Chemokines and CD4 T Cell Accumulation in Intestine

Migration of naïve and activated lymphocytes is regulated by the expression of various molecules such as chemokine receptors and ligands. CD69, the early activation marker of C-type lectin domain family, is also shown to regulate the lymphocyte migration by affecting their egress from the thymus and secondary lymphoid organs. Here, we aimed to investigate the role of CD69 in accumulation of CD4 T cells in intestine using murine models of inflammatory bowel disease. We found that genetic deletion of CD69 in mice increases the expression of the chemokines CCL-1, CXCL-10 and CCL-19 in CD4(+) T cells and/or CD4(-) cells. Efficient in vitro migration of CD69-deficient CD4 T cells toward the chemokine stimuli was the result of increased expression and/or affinity of chemokine receptors. In vivo CD69(-/-) CD4 T cells accumulate in the intestine in higher numbers than B6 CD4 T cells as observed in competitive homing assay, dextran sodium sulphate (DSS)-induced colitis and antigen-specific transfer colitis. In DSS colitis CD69(-/-) CD4 T cell accumulation in colonic lamina propria (cLP) was associated with increased expression of CCL-1, CXCL-10 and CCL-19 genes. Furthermore, treatment of DSS-administrated CD69(-/-) mice with the mixture of CCL-1, CXCL-10 and CCL-19 neutralizing Abs significantly decreased the histopathological signs of colitis. Transfer of OT-II×CD69(-/-) CD45RB(high) CD4 T cells into RAG(-/-) hosts induced CD4 T cell accumulation in cLP. This study showed CD69 as negative regulator of inflammatory responses in intestine as it decreases the expression of chemotactic receptors and ligands and reduces the accumulation of CD4 T cells in cLP during colitis.

A point mutation in cpsE renders Streptococcus pneumoniae nonencapsulated and enhances its growth, adherence and competence

BackgroundThe polysaccharide capsule is a major virulence factor of the important human pathogen Streptococcus pneumoniae. However, S. pneumoniae strains lacking capsule do occur.ResultsHere, we report a nasopharyngeal isolate of Streptococcus pneumoniae composed of a mixture of two phenotypes; one encapsulated (serotype 18C) and the other nonencapsulated, determined by serotyping, electron microscopy and fluorescence isothiocyanate dextran exclusion assay.By whole genome sequencing, we demonstrated that the phenotypes differ by a single nucleotide base pair in capsular gene cpsE (C to G change at gene position 1135) predicted to result in amino acid change from arginine to glycine at position 379, located in the cytoplasmic, enzymatically active, region of this transmembrane protein. This SNP is responsible for loss of capsule production as the phenotype is transferred with the capsule operon. The nonencapsulated variant is superior in growth in vitro and is also 117-fold more adherent to and more invasive into Detroit 562 human epithelial cells than the encapsulated variant.Expression of six competence pathway genes and one competence-associated gene was 11 to 34-fold higher in the nonencapsulated variant than the encapsulated and transformation frequency was 3.7-fold greater.ConclusionsWe identified a new single point mutation in capsule gene cpsE of a clinical S. pneumoniae serotype 18C isolate sufficient to cause loss of capsule expression resulting in the co-existence of the encapsulated and nonencapsulated phenotype. The mutation caused phenotypic changes in growth, adherence to epithelial cells and transformability. Mutation in capsule gene cpsE may be a way for S. pneumoniae to lose its capsule and increase its colonization potential.

Insight into the dynamic ligand exchange process involved in bipyridyl linked arene ruthenium metalla-rectangles

The dynamic ligand exchange behavior of cationic arene ruthenium metalla-rectangles of the type [(pcymene) 4Ru4(OOXOO)2(NXN)2]4+ (OOXOO ¼ oxalato, 2,5-dioxydo-1,4-benzoquinonato, 5,8-dioxydo-1,4-naphthoquinonato; NXN ¼ 4,40-bipyridine-H8, 4,40-bipyridine-D8) has been studied in solution. The robustness of the rectangular architecture has been evidenced by NMR and ESI mass spectrometry. Thermodynamic and kinetic aspects of the ligand exchange process have been explored using 1H/2D isotope labeling of the 4,40-bipyridine connectors. This study shows that ligand exchange does not proceed spontaneously for these metalla-assemblies, even at high temperature, unless an external stimulus is applied.

A novel universal DNA labeling and amplification system for rapid microarray-based detection of 117 antibiotic resistance genes in Gram-positive bacteria.

A rapid and simple DNA labeling system has been developed for disposable microarrays and has been validated for the detection of 117 antibiotic resistance genes abundant in Gram-positive bacteria. The DNA was fragmented and amplified using phi-29 polymerase and random primers with linkers. Labeling and further amplification were then performed by classic PCR amplification using biotinylated primers specific for the linkers. The microarray developed by Perreten et al. (Perreten, V., Vorlet-Fawer, L., Slickers, P., Ehricht, R., Kuhnert, P., Frey, J., 2005. Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria. J.Clin.Microbiol. 43, 2291-2302.) was improved by additional oligonucleotides. A total of 244 oligonucleotides (26 to 37 nucleotide length and with similar melting temperatures) were spotted on the microarray, including genes conferring resistance to clinically important antibiotic classes like β-lactams, macrolides, aminoglycosides, glycopeptides and tetracyclines. Each antibiotic resistance gene is represented by at least 2 oligonucleotides designed from consensus sequences of gene families. The specificity of the oligonucleotides and the quality of the amplification and labeling were verified by analysis of a collection of 65 strains belonging to 24 species. Association between genotype and phenotype was verified for 6 antibiotics using 77 Staphylococcus strains belonging to different species and revealed 95% test specificity and a 93% predictive value of a positive test. The DNA labeling and amplification is independent of the species and of the target genes and could be used for different types of microarrays. This system has also the advantage to detect several genes within one bacterium at once, like in Staphylococcus aureus strain BM3318, in which up to 15 genes were detected. This new microarray-based detection system offers a large potential for applications in clinical diagnostic, basic research, food safety and surveillance programs for antimicrobial resistance.

Covalent immobilisation of VEGF on plasma-coated electrospun scaffolds for tissue engineering applications.

Recent findings in the field of biomaterials and tissue engineering provide evidence that surface immobilised growth factors display enhanced stability and induce prolonged function. Cell response can be regulated by material properties and at the site of interest. To this end, we developed scaffolds with covalently bound vascular endothelial growth factor (VEGF) and evaluated their mitogenic effect on endothelial cells in vitro. Nano- (254±133 nm) or micro-fibrous (4.0±0.4 μm) poly(ɛ-caprolactone) (PCL) non-wovens were produced by electrospinning and coated in a radio frequency (RF) plasma process to induce an oxygen functional hydrocarbon layer. Implemented carboxylic acid groups were converted into amine-reactive esters and covalently coupled to VEGF by forming stable amide bonds (standard EDC/NHS chemistry). Substrates were analysed by X-ray photoelectron spectroscopy (XPS), enzyme-linked immuno-assays (ELISA) and immunohistochemistry (anti-VEGF antibody and VEGF-R2 binding). Depending on the reaction conditions, immobilised VEGF was present at 127±47 ng to 941±199 ng per substrate (6mm diameter; concentrations of 4.5 ng mm(-2) or 33.3 ng mm(-2), respectively). Immunohistochemistry provided evidence for biological integrity of immobilised VEGF. Endothelial cell number of primary endothelial cells or immortalised endothelial cells were significantly enhanced on VEGF-functionalised scaffolds compared to native PCL scaffolds. This indicates a sustained activity of immobilised VEGF over a culture period of nine days. We present a versatile method for the fabrication of growth factor-loaded scaffolds at specific concentrations.

Promising anchoring groups for single-molecule conductance measurements

The understanding of the charge transport through single molecule junctions is a prerequisite for the design and building of electronic circuits based on single molecule junctions. However, reliable and robust formation of such junctions is a challenging task to achieve. In this topical review, we present a systematic investigation of the anchoring group effect on single molecule junction conductance by employing two complementary techniques, namely scanning tunneling microscopy break junction (STM-BJ) and mechanically controllable break junction (MCBJ) techniques, based on the studies published in the literature and important results from our own work. We compared conductance studies for conventional anchoring groups described earlier with the molecular junctions formed through π-interactions with the electrode surface (Au, Pt, Ag) and we also summarized recent developments in the formation of highly conducting covalent Au–C σ-bonds using oligophenyleneethynylene (OPE) and an alkane molecular backbone. Specifically, we focus on the electron transport properties of diaryloligoyne, oligophenyleneethynylene (OPE) and/or alkane molecular junctions composed of several traditional anchoring groups, (dihydrobenzo[b]thiophene (BT), 5-benzothienyl analogue (BTh), thiol (SH), pyridyl (PY), amine (NH2), cyano (CN), methyl sulphide (SMe), nitro (NO2)) and other anchoring groups at the solid/liquid interface. The qualitative and quantitative comparison of the results obtained with different anchoring groups reveals structural and mechanistic details of the different types of single molecular junctions. The results reported in this prospective may serve as a guideline for the design and synthesis of molecular systems to be used in molecule-based electronic devices.