Development of Anti-LRRC15 Small Fragments for Imaging Purposes Using a Phage-Display ScFv Approach

The human leucine-rich repeat-containing protein 15 (LRRC15) is a membrane protein identified as a marker of CAF (cancer-associated fibroblast) cells whose overexpression is positively correlated with cancer grade and outcome. Nuclear molecular imaging (i.e., SPECT and PET) to track LRRC15 expression could be very useful in guiding further therapeutic strategies. In this study, we developed an ScFv mouse phage-display library to obtain small fragment antibodies against human LRRC15 for molecular imaging purposes. Mice were immunized with recombinant human LRRC15 (hLRRC15), and lymph node cells were harvested for ScFv (single-chain variable fragment) phage-display analysis. The built library was used for panning on cell lines with constitutive or induced expression after transfection. The choice of best candidates was performed by screening various other cell lines, using flow cytometry. The selected candidates were reformatted into Cys-ScFv or Cys-diabody by addition of cysteine, and cloned in mammalian expression vectors to obtain batches of small fragments that were further used in site-specific radiolabeling tests. The obtained library was 1.2 × 10⁷ cfu/µg with an insertion rate >95%. The two panning rounds performed on cells permittedenrichment of 2 × 10⁻³. Screening with flow cytometry allowed us to identify 28 specific hLRRC15 candidates. Among these, two also recognized murine LRCC15 and were reformatted into Cys-ScFv and Cys-diabody. They were expressed transiently in a mammalian system to obtain 1.0 to 4.5 mg of Cys fragments ready for bioconjugation and radiolabeling. Thus, in this paper, we demonstrate the relevance of the phage-display ScFv library approach for the fast-track development of small antibodies for imaging and/or immunotherapy purposes.     

Selective and uniform laser-induced failure of antireflection-coated LiNbO3surfaces

LiNbO3surfaces with different antireflection coatings and from different vendors were damaged with 1.06 μm 9.5 ns full width at half maximum laser pulses. By probing with a laser spot smaller than the separation of isolated surface defects, it was possible to separate uniform damage, which was characteristic of the coating itself, from premature defect-driven localized failures. Uniform failure modes were identified and thresholds extracted for each by multithreshold analysis. Frequency of defect damage was determined at several fluences much lower than required for uniform failure. It is likely that defect-driven selective failures reduce the apparent damage threshold of flood-loaded samples. Tentative identification of a selective damage threshold was made and correlated with large-spot multimode test results. Incorporation of these results into a product-improvement program has resulted in LiNbO3Q-switches with increased resistance to selective damage.     

Survey of liquefaction structures induced by recent moderate earthquakes

In April–May 1989, the eastern coastlands of Falcon State (Northwestern Venezuela) were affected by a seismic swarm of over 2000 weak shallow events located on the continental platform. Nevertheless two moderate earthquakes (mb 5.7 and 5.0) occurred during this swarm, [anicking inhabitants and producing scarce structural damage but frequent fissures, fractures and wall falls of house masonry in the mesoseismal area. The most relevant geological features observed in the mesoseismal area, as a consequence of these main events, are related to liquefaction of saturated sand-layers and are concentrated in the flood plains and delta of the Tocuyo river, located less than 20 km away from the epicentral area. Both shocks triggered liquefaction with ground acceleration values ranging between 0.1 and 0.3 g. The FUNVISIS Earth Sciences Workgroup carried out a prospect-pit survey across these features (sand boils and vent-fractures) to have a better understanding of this phenomenon and to evaluate the liquefaction potential of sand layers in the mesoseismal area. The liquefied material came to the surface from subsurficial silty-sand layers deposited in fluvio-deltaic environments and located between 0.2 and 4.0 m in depth. The pathways used by liquefied sands correspond to pre-existing crab burrows and soil fractures.     

A Study of Human Reactions to Emissions from Building Materials in Climate Chambers. Part II: VOC Measurements,Mouse Bioassay,and Decipol Evaluation in the 1–2 mg/m3 TVOC Range

Monitoring of human reactions to the emission of formaldehyde and volatile organic compounds (VOC) from four commonly used building materials was carried out. The building materials were: a painted gypsum board, a rubber floor, a nylon carpet, and a particle board with an acid-curing paint. The exposures were performed in climate chambers. The air quality was quantified on the decipol scale by a trained panel, measurements of formaldehyde and VOC being performed simultaneously. The irritating potency of the materials was measured by a mouse bioassay. The VOC measurements showed several malodorants and irritants. Some abundant VOC identified in the head-space analyses were absent in the climate chamber air. The rubber floor and the nylon carpet exhibited a marked increase in decipols compatible with a number of odorous VOC identified in the air. A high formaldehyde concentration (minimum 743μg/m³) was measured for the particle board coated with an acid-curing paint. This was not reflected by a corresponding relatively high decipol value but a long-lasting irritating potency was observed in the mouse bioassay. TVOC sampled on Tenax and expressed in mass per volume as well as in molar concentration, and decipol evaluation both have limitations and should be used with caution as indicators of (perceived) indoor air quality. Eye irritation expressed by means of the eye index reflecting the tear film quality index (comprised of break-up time, foam formation, thickness of the precorneal lipid layer of the tear film, and epithelial damage) was found to be insensitive to formaldehyde and a VOC mixture but sensitive to TVOC concentrations of 1–2 mg/m³. Lipophilic VOC may be the cause of reduced tear film quality by destabilization of the lipid multilayer of the tear film.     

Experimental right ventricle to pulmonary artery discontinuity: outcome of polyurethane valved conduits

OBJECTIVE: The ideal substitute for the treatment of ventricle-pulmonary artery discontinuity remains a topic of controversy, because of calcifications and degeneration of biologic substitutes leading to subsequent reoperations. Because polyurethane valves used in ventricular assist devices show a satisfactory biocompatibility, the aim of this study was to evaluate a valved conduit composed of a Dacron graft incorporating a trileaflet 25 mm polyurethane valve. METHODS: The conduit was implanted between the right ventricle and the main pulmonary artery in adult sheep, with ligation of the proximal pulmonary artery. The animals received no medications. Serial hemodynamic data were collected at the time of implantation and at postoperative intervals of 6 and 12 months. RESULTS: The peak pressure gradient across the valve increased significantly between implantation (0.17 +/- 5.6 mm Hg) and 6 months after operation (7.3 +/- 3 mm Hg, p = 0.0007) and remained stable thereafter (6.7 +/- 3 mm Hg at 12 months), whereas the cardiac output remained unchanged (4.6 +/- 0.6 L/min at implantation, 4 +/- 0.6 L/min at 6 months, and 3.9 +/- 1.1 L/min at 12 months). At the completion of the study, valve samples were processed and vapor coated with carbon for microscopic examination. There was one instance of nonadherent thrombus formation inside a cusp but no structural failures. The other valves were free of calcium deposits and no significant amounts of phosphorus could be detected by scanning electron microscopy and energy dispersive spectrometry. CONCLUSIONS: These data demonstrate the good hemodynamic performance, low thrombogenicity, and acceptable durability of the polyurethane valves implanted in the right side of the heart in a chronic sheep model.     

A quantitative treatment of oscillatory phenomena in coarse-grained ion-exchanger membranes

The treatment developed in this text concerns the ion flux, volume flux and electric current through the highly porous ion-exchanger membrane of the Teorell membrane oscillator. It is based on the concept that inside the membrane exists a concentration profile of defined shape depending mainly on the volume flux caused by the hydrostatic pressure and the electro-osmotic effect of the membrane potential within the reach of the electric charge of the ion-exchanger grains forming the membrane texture.In particular it is shown that under conditions of zero volume flux the effective fixed charge responsible for the electro-osmotic driving force can be determined directly by measuring the hydrostatic pressure and its compensating electric potential. Like the electro-osmotic effect, also the derived flux equations proved to be independent of the special feature of the grain texture.The simultaneous differential equations describing the temporal (oscillatory) behaviour of the Teorell membrane system are derived on the basis of these flux equations leading to analog computer solutions which agree well with the experimental data.