We have analyzed the proliferative and differentiation potential of human ocular keratinocytes. Holoclones, meroclones, and paraclones, previously identified in skin, constitute also the proliferative compartment of the ocular epithelium. Ocular holoclones have the expected properties of stem cells, while transient amplifying cells have variable proliferative potential. Corneal stem cells are segregated in the limbus, while conjunctival stem cells are uniformly distributed in bulbar and forniceal conjunctiva. Conjunctival keratinocytes and goblet cells derive from a common bipotent progenitor. Goblet cells were found in cultures of transient amplifying cells, suggesting that commitment for goblet cell differentiation can occur late in the life of a single conjunctival clone. We found that conjunctival keratinocytes with high proliferative capacity give rise to goblet cells at least twice in their life and, more importantly, at rather precise times of their life history, namely at 45–50 cell doublings and at ∼15 cell doublings before senescence. Thus, the decision of conjunctival keratinocytes to differentiate into goblet cells appears to be dependent upon an intrinsic “cell doubling clock.” These data open new perspectives in the surgical treatment of severe defects of the anterior ocular surface with autologous cultured conjunctival epithelium.

Ocular burns cause depletion of limbal stem cells, which leads to corneal opacification and visual loss. Autologous cultured epithelial cells can restore damaged corneas, but this technology is still developing. We sought to establish a culture system that allows preservation of limbal stem cells and preparation of manageable epithelial sheets and to investigate whether such cultures can permanently restore total limbal stem cell deficiency. We selected a homogeneous group of patients whose limbal cell deficiency was evaluated by scoring the gravity of the clinical picture and the keratin expression pattern. Stem cells, obtained from the limbus of the contralateral eye, were cultivated onto a fibrin substrate and their preservation was evaluated by clonal analysis. Fibrin cultures were grafted onto damaged corneas. Fibrin-cultured limbal stem cells were successful in 14 of 18 patients. Re-epithelialization occurred within the first week. Inflammation and vascularization regressed within the first 3–4 weeks. By the first month, the corneal surface was covered by a transparent, normal-looking epithelium. At 12–27 months follow-up, corneal surfaces were clinically and cytologically stable. Three patients had a penetrating keratoplasty approximately 1 year after restoration of their corneal surface. Their visual acuity improved from light perception or counting fingers to 0.8–1.0. Preservation of limbal stem cells in culture gives new perspectives on the treatment of ocular disorders characterized by complete limbal stem cell deficiency. The multicenter nature of this study and the handiness and ease of long-distance transportation of the fibrin-cultured epithelial sheets suggest that this technology can now be widely applied.

Nerve growth factor (NGF) serum levels were measured in 49 patients with asthma and/or rhinoconjunctivitis and/or urticaria-angioedema. Clinical and biochemical parameters, such as bronchial reactivity, total and specific serum IgE levels, and circulating eosinophil cationic protein levels, were evaluated in relation to NGF values in asthma patients. NGF was significantly increased in the 42 allergic (skin-test- or radioallergosorbent-test-positive) subjects (49.7 +/- 28.8 pg/ml) versus the 18 matched controls (3.8 +/- 1.7 pg/ml; P < 0.001). NGF levels in allergic patients with asthma, rhinoconjunctivitis, and urticaria-angioedema were 132.1 +/- 90.8, 17.6 +/- 6.1, and 7.6 +/- 1.8 pg/ml (P < 0.001, P < 0.002, and P < 0.05 versus controls), respectively. Patients with more than one allergic disease had higher NGF serum values than those with a single disease. When asthma patients were considered as a group, NGF serum values (87.6 +/- 59.8 pg/ml) were still significantly higher than those of control groups (P < 0.001), but allergic asthma patients had elevated NGF serum levels compared with nonallergic asthma patients (132.1 +/- 90.8 versus 4.9 +/- 2.9 pg/ml; P < 0.001). NGF serum levels correlate to total IgE serum values (rho = 0.43; P < 0.02). The highest NGF values were found in patients with severe allergic asthma, a high degree of bronchial hyperreactivity, and high total IgE and eosinophil cationic protein serum levels. This study represents the first observation (that we know of) that NGF is increased in human allergic inflammatory diseases and asthma.