Background/aims-Deturgescence of the corneal stroma is controlled by the pumping action of the endothelial layer and can be monitored by measurement of central corneal thickness (pachymetry). Loss or damage of endothelial cells leads to an increase in corneal thickness, which may ultimately induce corneal decompensation and loss of vision. Little is known about the eVect of moderate reductions in endothelial cell number on the thickness of the corneal stroma. This study aimed to investigate this matter further using patients who had incurred moderate decreases in their endothelial cell counts as a result of cataract surgery. Methods-Central corneal thickness was measured 1 day before surgery, 1 day after surgery, and again at 3 months or 1 year. Endothelial cell counts were also performed 1 day before surgery and thereafter at 3 months or 1 year after surgery. The relationship between these two parameters was assessed statistically. Precise measurements of central corneal thickness were made by optical low coherence reflectometry. For comparative purposes, this parameter was also determined by ultrasonic pachymetry. Central corneal endothelial cell numerical density was estimated on photomicrographs taken with a specular microscope. Results-All patients had significant postoperative corneal swelling on the day after surgery; preoperative values were restored by 3 and 12 months, even though significant endothelial cell losses had occurred. No correlation existed between central corneal thickness and central corneal endothelial cell numerical density. Measurements estimated by ultrasonic pachymetry were more variable and significantly higher than those determined by optical low coherence reflectometry. Conclusion-As long as the numerical density of the corneal endothelial cells does not fall below the physiological threshold, a moderate decrease in this parameter does not compromise the pumping activity of the layer as a whole. (Br J Ophthalmol 2001;85:18-20)

Background/aims-Recent studies have revealed patients with ocular hypertension to have thicker than normal central corneas and those with normal tension glaucoma to have thinner than normal ones, as determined by ultrasonic pachymetry. Since corneal thickness measurements and applanation tonometric estimates of intraocular pressure (IOP) correlate positively, monitoring of the former parameter have served as the basis for adjusting readings pertaining to the latter, with the consequence that many patients have had to be reclassified. With a view to validating these pachymetric studies, the central corneal thickness was determined in patients with normal tension glaucoma, primary open angle glaucoma, pseudoexfoliation glaucoma, or ocular hypertension, as well as that of normal subjects, using optical low coherence reflectometry, which is a new and more precise method than ultrasonic pachymetry. Methods-34 patients with normal tension glaucoma, 20 with primary open angle glaucoma, 13 with pseudoexfoliation glaucoma, and 12 with ocular hypertension, together with 21 control subjects, were included in this observational, concurrent case-control study. One eye per individual was randomly selected for investigation. IOP was measured by Goldmann applanation tonometry and central corneal thickness by optical low coherence reflectometry. Results-Central corneal thickness was significantly higher (p <0.001) in patients with ocular hypertension than in normal individuals or in subjects with either normal tension glaucoma, primary open angle glaucoma, or pseudoexfoliation glaucoma, there being no significant differences between the latter four groups. Patients with ocular hypertension were also significantly younger (p <0.003) than those within any of the three glaucomatous groups. Conclusion-This study confirms that a significant number of patients with ocular hypertension have normal IOPs after the appropriate adjustments have been made for deviations from normal in their central corneal thickness. The accurate measurement of this latter parameter is important not only for individual patient care, in permitting more precise estimations of IOP, but also for clinical studies, in assuring a more reliable classification of subjects. (Br J Ophthalmol 2001;85:792-795)

This is the first study showing the maintenance of adult human RPE-specific cell differentiation and characteristics in vitro after primary culture and after cryopreservation using improved cell culture methods. The optimization and quality control of cell culture is an important prerequisite for successful cell transplantation.

Background/aims-Bacterial endotoxin is a potent inflammatory stimulator, the local and systemic responses thereby elicited being mediated via the release of cytokines from diverse cell types. Under physiological conditions, the corneal endothelium is protected from these toxins by the epithelial and vascular barriers, but in organ culture these safeguards are no longer operative, and such substances will therefore have ready access to this cell layer. The consequences of such exposure may take the form of overt damage to the endothelium and/or a more discreet influence on the cornea's immunological status, the eVects of which may be realised only after transplantation, by its poor performance. The media bathing organ cultured donor corneas were monitored for the presence of various cytokine mediators of the inflammatory response before and after incubation with endotoxin, and these data compared with those pertaining to endothelial cell morphology and numerical density. Methods-Six pairs of fellow donor corneas were cultured for an initial equilibration period of 10 days and then transferred to fresh medium; thereafter, one of each pair was incubated in the absence, and the other in the presence, of endotoxin (50 µg/ml ≡ 25 000 units/ml), and culturing continued for a further 10 days. Samples of medium were withdrawn at regular intervals throughout the 20 days and screened for the cytokines IL-1, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, GM-CSF, and TNF by ELISA; endothelial cell morphology and area density were assessed on days 0, 10, and 20. Results-Spiking of organ culture media with endotoxin led to a substantial increase in the level of IL-8, and a smaller one in that of IL-6, but none of the other cytokines were detected. In five of the six stimulated corneas, these changes coincided with an increased incidence of endothelial cell loss, compared with that incurred by the fellow control, and the surviving population also evinced signs of degeneration not seen in the latter. Conclusion-Endotoxin induced increases in the levels of IL-6 and IL-8 appear to be correlated with endothelial cell loss. Since no adverse eVects of this toxin on long term cultured monolayers of human corneal endothelial cells have been previously observed, the damage incurred in corneal organ culture may well be attributable to the influence of cytokines produced by other corneal cells or a non-intrinsic (passenger) cell population, such as macrophages, Langerhans cells, or lymphocytes present under these latter conditions.

Cytokine-Induced (CIK) cells represent an attractive approach for cell-based immunotherapy, as they show several advantages compared with other strategies. Here we describe an original serum-free protocol for CIK cell expansion that employs G-Rex devices and compare the resulting growth, viability, phenotypic profile and cytotoxic activity with conventional culture in tissue flasks. CIK cells were obtained from buffy coats, seeded in parallel in G-Rex and tissue flasks, and stimulated with clinical-grade IFN-g, anti-CD3 antibody and IL-2. G-Rex led to large numbers of CIK cells, with a minimal need for technical interventions, thus reducing the time and costs of culture manipulation. CIK cells generated in G-Rex showed a less differentiated phenotype, with a significantly higher expression of naive-associated markers such as CD62L, CD45RA and CCR7, which correlates with a remarkable expansion potential in culture and could lead to longer persistence and a more sustained anti-tumor response in vivo. The described procedure can be easily translated to large-scale production under Good Manufacturing Practice. Overall, this protocol has strong advantages over existing procedures, as it allows easier, time-saving and cost-effective production of CIK effector cells, fostering their clinical application.