Alessandro Lambiase

Autologous fibrin-cultured limbal stem cells permanently restore the corneal surface of patients with total limbal stem cell deficiency.

BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Topical treatment with nerve growth factor for corneal neurotrophic ulcers

BACKGROUND Corneal neurotrophic ulcers associated with impairment of sensory innervation of the cornea may lead to loss of vision, and there is no effective treatment for these ulcers. We evaluated the effects of nerve growth factor in patients with this disorder. METHODS Twelve patients (14 eyes) with severe neurotrophic corneal ulcers associated with corneal anesthesia were treated with topical nerve growth factor 10 times daily for two days and then 6 times daily until the ulcers healed. Treatment continued for 2 weeks after the ulcers healed, and the patients were then followed for up to 12 months. The evolution of the corneal disease during treatment and follow-up was evaluated by slit-lamp examination, photography, fluorescein-dye testing, and tests of corneal sensitivity and best corrected visual acuity. RESULTS Corneal healing began 2 to 14 days after the initiation of treatment with nerve growth factor, and all patients had complete healing of their corneal ulcers after 10 days to 6 weeks of treatment. Corneal sensitivity improved in 13 eyes, and returned to normal in 2 of the 13 eyes. Corneal integrity and sensitivity were maintained during the follow-up period (range, 3 to 12 months). Best corrected visual acuity increased progressively during treatment and follow-up in all patients. There were no systemic or local side effects of treatment. CONCLUSIONS In this preliminary, uncontrolled study, topically applied exogenous nerve growth factor restored corneal integrity in patients with corneal neurotrophic ulcers.

Vernal keratoconjunctivitis revisited: A case series of 195 patients with long-term followup

OBJECTIVE This study aimed at revisiting vernal keratoconjunctivitis (VKC) on the basis of anamnestic, clinical, immunologic, histopathologic, and followup data of 195 patients. DESIGN Retrospective noncomparative case series. PARTICIPANTS One hundred and ninety-five patients with VKC. METHODS Clinical evaluation and outcome in 151 of 195 patients with a median followup of 47 months. Evaluation was by telephone survey in 69 patients. MAIN OUTCOME MEASURES (1) Demographic, clinical, and immunologic features of VKC and their influence on the course of the disease; (2) conjunctival and corneal complications and efficacy of treatment observed during the followup period. RESULTS VKC is a chronic disease. More than 60% of patients had repeated recurrences all year round. Males had an earlier presentation of symptoms than females and the male/female ratio decreased with age. Major (greater than 80%) and minor (up to 80%) diagnostic criteria were defined for clinical signs and symptoms of the disease. Negative skin test or radioallergosorbent test was present in approximately 50% of patients, whereas eosinophil infiltration was a constant histopathologic finding. A marked conjunctival sensitivity to nonspecific stimuli was noted in more than one third of patients. In 6% of cases, a reduction of visual acuity resulted from corneal scarring, and in 2% of patients, steroid-induced glaucoma was observed. The large size of giant papillae indicates poor prognosis for the persistence of the disease and its evolution into a chronic, perennial condition. CONCLUSIONS VKC is a chronic eosinophilic disease of the ocular surface involving IgE, non IgE-mediated mechanisms, and age-sex-related influences. Although the disease has a good prognosis, severe visual impairments may result from long-standing inflammation.

Human CD4+ T cell clones produce and release nerve growth factor and express high-affinity nerve growth factor receptors

BACKGROUND Increasing evidence shows that nerve growth factor (NGF) plays a role in the complex and fascinating linkage between the nervous and the immune systems due to its ability to modulate functions of several inflammatory cells. OBJECTIVE To investigate NGF receptor expression and NGF production and release by human CD4+ cells clones, which have primary relevance in modulating inflammatory events through their different subsets of functional phenotypes. METHODS The expression of NGF and a transmembrane tyrosine kinase (TrkA) was evaluated by immunohistochemistry and flow cytometry analysis in five T(H0), six T(H1), and five T(H2) cell clones derived from human circulating mononuclear blood cells. Moreover, the amount of NGF protein was assessed by measuring the NGF levels in culture supernatants of the T cell clones before stimulation and 48 hours after phytohemagglutinin (PHA) activation by use of an immunoenzymatic assay. RESULTS Our data have shown that in unstimulated conditions, human CD4+ T cell clones express both immunoreactivity for NGF and the TrkA NGF receptor irrespective of their cytokine profile. Moreover, T(H1) and T(H2) clones, but not T(H0) clones, secrete NGF in basal conditions. PHA activation induces NGF secretion in T(H0) clones and a significant increase of NGF levels in T(H2) (p < 0.05), but not in T(H1) culture supernatants. CONCLUSIONS Results obtained represent the first evidence of TrkA expression and NGF production and release in human CD4+ cell clones and suggest a possible functional role of NGF in modulating the immune and inflammatory network.

Topical treatment with nerve growth factor for neurotrophic keratitis

OBJECTIVE To evaluate the efficacy of nerve growth factor (NGF) in patients with neurotrophic keratitis. DESIGN Prospective, noncomparative, interventional case series. PARTICIPANTS Forty-five eyes of 43 consecutive patients with moderate (stage 2, n = 17) to severe (stage 3, n = 28) neurotrophic keratitis unresponsive to other nonsurgical therapies. METHODS After a 10-day washout with preservative-free artificial tears, 45 eyes with neurotrophic keratitis received murine NGF (200 microg/ml) every 2 hours for 2 days followed by one drop six times daily until the ulcer healed. A maintenance dose of one drop NGF (100 microg/ml) was administered four times daily for the 2 weeks subsequent to ulcer healing. MAIN OUTCOME MEASURES Size and depth of the ulcer or the epithelial defect, corneal sensitivity, best corrected visual acuity, side effects, and relapse of the disease in the follow-up period. RESULTS All patients had a complete resolution of the persistent epithelial defect (with or without an ulcer) after 12 days to 6 weeks of treatment with NGF. Patients affected by both stages of the disease demonstrated both improved corneal sensitivity and visual acuity (P<0.001). No significant differences were observed in the time to complete corneal healing between stage 2 and stage 3 patients. Hyperemia and ocular and periocular pain were side effects reported during the first days of treatment. No relapse of the disease was observed during the follow-up period, with the exception of three patients with trigeminal nerve resection, who required a single retreatment. CONCLUSIONS Nerve growth factor eye drops improved corneal sensitivity and promoted corneal epithelial healing in both moderate and severe neurotrophic keratitis. Although performed in an uncontrolled and nonrandomized series of patients, this therapy shows promise for the restoration of ocular surface integrity and visual function in neurotrophic corneal disease.

Experimental and clinical evidence of neuroprotection by nerve growth factor eye drops: Implications for glaucoma

Elevated intraocular pressure (IOP) in glaucoma causes loss of retinal ganglion cells (RGCs) and damage to the optic nerve. Although IOP is controlled pharmacologically, no treatment is available to restore retinal and optic nerve function. We evaluated the effects of NGF eye drops in a rat model of glaucoma. We also treated 3 patients with progressive visual field defects despite IOP control. Glaucoma was induced in rats through injection of hypertonic saline into the episcleral vein. Initially, 2 doses of NGF (100 and 200 μg/mL) were tested on 24 rats, and the higher dose was found to be more effective. Glaucoma was then induced in an additional 36 rats: half untreated and half treated with 200 μg/mL NGF QID for 7 weeks. Apoptosis/survival of RGCs was evaluated by histological, biochemical, and molecular analysis. Three patients with advanced glaucoma underwent psychofunctional and electrofunctional tests at baseline, after 3 months of NGF eye drops, and after 3 months of follow-up. Seven weeks of elevated IOP caused RGC degeneration resulting in 40% cell death. Significantly less RGC loss was observed with NGF treatment (2,530 ± 121 vs. 1,850 ± 156 RGCs/mm2) associated with inhibition of cell death by apoptosis. Patients treated with NGF demonstrated long lasting improvements in visual field, optic nerve function, contrast sensitivity, and visual acuity. NGF exerted neuroprotective effects, inhibiting apoptosis of RGCs in animals with glaucoma. In 3 patients with advanced glaucoma, treatment with topical NGF improved all parameters of visual function. These results may open therapeutic perspectives for glaucoma and other neurodegenerative diseases.

Nerve growth factor: an important molecule in allergic inflammation and tissue remodelling

Background: Several studies provide evidence that nerve growth factor (NGF) has an expanding role in neuroimmune interactions. Methods: We review our data on circulation levels of NGF in allergic diseases as well as on the relationships between this neurotrophin and primary and secondary effector cells of allergic inflammation. Results: In vernal keratoconjunctivitis, a close relationship exists between the increased NGF plasma values and the number of mast cells infiltrating the conjunctiva. NGF serum values are also increased in other allergic diseases and asthma, and are related to the severity of the inflammatory process and disease. Human CD4+ T cell clones (preferentially of activated Th2 type) produce and release NGF, and express high–affinity NGF receptors. NGF is preformed in and can act on human peripheral blood eosinophils to preferentially release inflammatory mediators. Immunoreactivity for high affinity NGF receptors is present both in basal epithelial cells and in the inflamed stroma of the allergic conjunctiva. Topical administration of NGF results in a complete healing of neurotophic corneal ulcers in man, thus suggesting a profound effect of NGF on human fibroblasts and extracellular matrix. Conclusion: Data presented suggest that NGF is an important molecule in allergic inflammation and tissue remodelling occurring in allergic diseases.

Effect of topical application of nerve-growth factor on pressure ulcers

Pressure ulcer is a frequent problem in frail older people, with serious consequences and high costs. Topical application of nerve growth factor promotes skin ulcer healing.

Management of neurotrophic keratopathy.

Neurotrophic keratopathy is a degenerative corneal disease caused by an impairment of corneal sensitivity. Lack of the sensory nerves trophic effect is responsible for the impairment in corneal healing and for the changes on the ocular surface that lead to corneal epithelial deficit, ulcer, and perforation. The etiology and recent advances in understanding of the pathogenetic mechanisms of neurotrophic keratopathy are reviewed here. An accurate history and a clinical examination that covers the function of cranial nerves often identify the cause of the disease. Clinical features and guidelines for the differential diagnosis and treatment are presented. Specific medical and surgical treatments, selected on the basis of clinical staging of the disease, can often halt disease progression. Future developments in medical treatment, including the use of neuropeptide and growth factors, are discussed. The identification of corneal anesthesia associated with an epithelial defect allows appropriate treatment and prevention of progression to stromal lysis and perforation.

Nerve growth factor delays retinal degeneration in C3H mice

Abstract• Background: The aim of the present study was to investigate the biological role of nerve growth factor (NGF) on retinal degeneration in the C3H mouse strain. This strain is characterized by a single gene mutation (rd) which leads to photoreceptor degeneration resembling human retinitis pigmentosa. • Methods: Neural retinas from 1- to 25 day-old C3H mice were dissected from outer ocular tissues, dissociated in cell suspension, stained with a vital dye and counted in a hemocytometer. For in vivo study, NGF was injected into the intraocular or retro-ocular area, and at the end of the treatment the mice were killed. The eyes were enucleated, fixed and cut by cryostat into 14-μm serial sections. The serial sections were stained with hematoxylin-eosin and the outer nuclear layer (ONL) was measured using a computerized image analysis system. • Results: An intraocular injection of NGF, or repeated retro-ocular injections, induced a significant increase in ONL thickness compared to controls. • Conclusion: Our data show that NGF inhibits retinal degeneration in C3H mice. The mechanism(s) underlying the protective action of NGF against retinal cell death remains to be established.