This study reports a new method for fabricating bioengineered human corneal endothelial cell sheets suitable for ocular surgery and repair. We have initially cultured human corneal endothelial cells on type IV collagen-coated dishes and, after several passages, expanded cells were then seeded onto novel temperature-responsive culture dishes. Four weeks after reaching confluence, these cultured endothelial cells were harvested as intact monolayer cell sheets by simple temperature reduction without enzymatic treatment. Scanning electron microscopy indicated that these cells were primarily hexagonal with numerous microvilli and cilia, similar to the native corneal endothelium. The Na+, K+-ATPase pump sites were located at the cell borders as in vivo. Moreover, cell densities and numbers of pump sites were identical to those of in vivo human corneal endothelium under optimized conditions. A 3H-ouabain binding analysis demonstrated a linear proportionality for cell pump density between confluent cell densities of 575 cells/mm2 and 3070 cells/mm2. We also confirmed Na+, K+-ATPase activity in the sheets in vitro. Xenograft transplantation results showed that the fabricated sheets retain their function of maintaining proper stromal hydration in vivo. We have established a regimen to culture and proliferate human corneal endothelial cells and fabricate endothelial sheets ex vivo morphologically and functionally similar to the native corneal endothelium. Our results support the value of harvested cell sheets for clinical applications in ocular reconstructive surgery in patients with ocular endothelial decompensation.

The immunoreceptor tyrosine-based activation motif (ITAM) is a highly conserved region in the cytoplasmic domain of signaling chains and receptors and is a critical mediator of intracellular signals. ITAM-mediated signals depend on the Syk or zeta-associated protein of 70 kDa tyrosine kinases, and ITAM signaling is required for the differentiation and function of B and T cells in adaptive immunity. ITAM-dependent receptors also regulate the function of innate immune cells, including natural killer cells, and myeloid-derived cells such as macrophages, neutrophils, dendritic cells, and mast cells. Myeloid lineage cells also include osteoclasts (OCLs), the cells required for bone resorption, and recent studies show a critical role for the ITAM-containing adapter proteins DAP12 and the FcRgamma chain (Fcepsilon receptor I gamma chain) in OCL differentiation. Mice deficient in both the DAP12 and FcRgamma ITAM-bearing adapters are significantly osteopetrotic with a severe defect in OCL differentiation, demonstrating the requirement for ITAM signals in bone and further implicating this pathway in the development of highly specialized cell functions in hematopoietic cells. Regulation of osteoclastogenesis by ITAM-dependent receptors suggests that OCLs, similar to related myeloid cells, are tightly controlled by arrays of receptors that allow them to sense and respond to their local microenvironment like other innate immune cells.

OBJECTIVE

We sought to determine whether nitrogen dioxide (NO2) can enhance airway inflammation after allergen challenge in asthmatic subjects.

METHODS

Fifteen house-dust-mite (HDM)-sensitive asthmatic subjects were exposed for 3 hours to filtered air or 0.4 ppm NO2, followed by inhalational challenge with HDM allergen. Markers of inflammation were measured in sputum at 6 hours and 26 hours after allergen challenge.

RESULTS

After exposure to NO2, eosinophil concentration decreased significantly in the 6-hour postallergen sputum. No significant NO2-related difference was observed for other variables.

CONCLUSIONS

Our results suggest that, in most asthmatic individuals, multi-hour exposure to a high ambient concentration of NO2 does not enhance the inflammatory response to subsequent inhaled allergen as assessed by cell distribution in induced sputum. Because the decrease in airway eosinophils has been reported in previous animal studies, future research should be directed toward the mechanism of this effect.