Silica-induced NLRP3 inflammasome activation in vitro and in rat lungs.

RATIONALE: Mineral particles in the lung cause inflammation and silicosis. In myeloid and bronchial epithelial cells the inflammasome plays a role in responses to crystalline silica. Thioredoxin (TRX) and its inhibitory protein TRX-interacting protein link oxidative stress with inflammasome activation. We investigated inflammasome activation by crystalline silica polymorphs and modulation by TRX in vitro, as well as its localization and the importance of silica surface reactivity in rats. METHODS: We exposed bronchial epithelial cells and differentiated macrophages to silica polymorphs quartz and cristobalite and measured caspase-1 activity as well as the release of IL-1ß, bFGF and HMGB1; including after TRX overexpression or treatment with recombinant TRX. Rats were intratracheally instilled with vehicle control, Dörentruper quartz (DQ12) or DQ12 coated with polyvinylpyridine N-oxide. At days 3, 7, 28, 90, 180 and 360 five animals per treatment group were sacrificed. Hallmarks of silicosis were assessed with Haematoxylin-eosin and Sirius Red stainings. Caspase-1 activity in the bronchoalveolar lavage and caspase-1 and IL-1ß localization in lung tissue were determined using Western blot and immunohistochemistry (IHC). RESULTS: Silica polymorphs triggered secretion of IL-1ß, bFGF and HMGB1 in a surface reactivity dependent manner. Inflammasome readouts linked with caspase-1 enzymatic activity were attenuated by TRX overexpression or treatment. At day 3 and 7 increased caspase-1 activity was detected in BALF of the DQ12 group and increased levels of caspase-1 and IL-1ß were observed with IHC in the DQ12 group compared to controls. DQ12 exposure revealed silicotic nodules at 180 and 360 days. Particle surface modification markedly attenuated the grade of inflammation and lymphocyte influx and attenuated the level of inflammasome activation, indicating that the development of silicosis and inflammasome activation is determined by crystalline silica surface reactivity. CONCLUSION: Our novel data indicate the pivotal role of surface reactivity of crystalline silica to activate the inflammasome in cultures of both epithelial cells and macrophages. Inhibitory capacity of the antioxidant TRX to inflammasome activation was evidenced. DQ12 quartz exposure induced acute and chronic functional activation of the inflammasome in the heterogeneous cell populations of the lung in associated with its crystalline surface reactivity.

Dose-response and threshold analysis of tumor prevalence after intratracheal instillation of six types of low- and high-surface-area particles in a chronic rat experiment.

Over a period of 20 years, rat experiments have consistently shown tumorigenic responses after exposure to poorly soluble low-toxicity particles (PSP). We performed a rigorous dose threshold analysis of a large previous rat study. Seven hundred and nine rats were intratracheally exposed to five different PSP: carbon black and titanium dioxide of low and high surface area, diesel emission particles (low surface area), and one soluble dust (amorphous silica, high surface area), at varying instilled total mass doses ranging from 3.0 mg to 120 mg. A multivariable Cox model was applied to analyse lung tumor prevalence. The model was extended by a dose threshold or a dose saturation parameter. This statistical approach, which is new in animal studies, showed no better fit when using surface area or volume as dose metrics but found significantly higher tumor prevalence in animals instilled with high-surface-area dust particles. Interestingly, a dose threshold of about 10 mg mass dose (0.95 CI: 5 mg to 15 mg) emerged from our calculations. In addition, our statistical analysis demonstrated that tumor prevalence is saturated beyond 20 mg mass dose. In summary, our analyses showed that these data are compatible with earlier observations that high-surface-area particles induce more lung tumors and support the concept of a dose threshold for lung tumor after PSP exposures in the rat. However, collinearities in the data (particle type and dose were correlated by design) and the saturation phenomenon (506 out of 709 rats were exposed above the estimated saturation dose) limit generalization of these findings.

Effects of ciprofloxacin on joint cartilage in immature dogs immediately after dosing and after a 5-month treatment-free period.

A study in young beagle dogs was performed in which the animals were treated for 2 weeks with ciprofloxacin at oral doses of 0, 10, 30 or 90 mg/kg per day. Immediately after treatment half of the number of animals were killed and all weight-bearing joints were subject to a thorough gross and histopathological investigation, including special staining of the cartilage matrix, and immunohistochemistry as well as electron microscopy. The remaining animals were maintained for an additional 5-months treatment-free period before being killed. Again, all weight-bearing joints were subject to a thorough gross and histopathological investigation. After 14 days of treatment with ciprofloxacin, oral doses of 30 and 90 mg/kg induced the characteristic arthropathy (blisters, erosions) in juvenile beagle dogs. As expected the lesions persisted while the animals were growing. In contrast, and to our knowledge demonstrated for the first time, an oral dose of 10 mg/kg ciprofloxacin did not induce joint lesions after short-term treatment in juvenile beagle dogs and was also not associated with arthrotoxicity when the dogs became older.

Intrastromal refractive surgery with ultrashort laser pulses: in vivo study on the rabbit eye.

BACKGROUND: Femtosecond (fs) laser pulses may offer new possibilities in the field of refractive surgery, especially when using the laser as a microkeratome. By induction of nonlinear absorption processes the laser can be used to perform intrastromal cuts. The conventional microkeratome, associated with numerous potential side effects, can possibly be replaced. Furthermore, refractive lenticules can be prepared within the stroma and removed in a single-step operation. METHODS: In 10 rabbits, cuts were made to create both a lamellar flap and an intrastromal refractive lenticule. The flap was lifted, the lenticule was extracted and, finally, the flap was repositioned (intrastromal laser keratomileusis, ILK). The corneal samples were collected up to 120 days after treatment and processed for histopathological analysis. RESULTS: All flaps could be opened and prepared lenticules could be extracted in one piece by the surgeon. The treated corneas developed a mild wound healing reaction, comparable to that known from excimer laser in situ keratomileusis (LASIK) studies. The wound healing was restricted to the flap-stroma interface, most pronounced at the periphery of the flaps. CONCLUSIONS: The use of the fs-laser offers new possibilities in preparation of corneal flaps, possibly providing advantages over conventional microkeratomes. Furthermore, the fs-laser has the potential to create intrastromal refractive lenticules for complete refractive procedures (ILK).

Function of the mammalian oviductal sperm reservoir.

Sperm are stored in the isthmic region of the oviduct under conditions that maintain sperm viability and suppress motility. This region is also the site in which essential steps of the capacitation process are coordinated with the appearance of the ovulated egg. The influx of Ca(2+) and phosphorylation of sperm proteins are features of the ongoing capacitation process. Using a cell-culture system of oviductal epithelial cells, it was found that sperm bound to the epithelial cells showed a reduced Ca(2+) uptake and almost no tyrosine phosphorylation as shown by indirect immunofluorescence. Furthermore, sperm viability, measured as membrane integrity with propidium iodide, is significantly prolonged as compared to sperm in suspension. The formation of the sperm reservoir appears to be mediated by carbohydrate-protein interaction. In the pig, it has been found that mannosyl-oligosaccharides exposed by the epithelial cells are high-affinity ligands for sperm-associated lectins. Ovalbumin and mannopentaose are effective inhibitors of sperm binding to explants of oviductal epithelium. Spermadhesins, a new class of animal lectins and the major secretory products of the porcine seminal vesicle, associate with the sperm surface at ejaculation and are candidate molecules for the receptors of the epithelial carbohydrates.