Claudio Macaluso

Biological parameters determining the clinical outcome of autologous cultures of limbal stem cells

AIM Limbal cultures restore the corneal epithelium in patients with ocular burns. We investigated the biological parameters instrumental for their clinical success. METHODS We report a long-term multicenter prospective study on 152 patients carrying corneal destruction due to severe ocular burns, treated with autologous limbal cells cultured on fibrin and clinical-grade 3T3-J2 feeder cells. Clinical results were statistically evaluated both by parametric and nonparametric methods. RESULTS Clinical outcomes were scored as full success, partial success and failure in 66.05, 19.14 and 14.81% of eyes, respectively. The total number of clonogenic cells, colony size, growth rate and presence of conjunctival cells could not predict clinical results. Instead, the clinical data provided conclusive evidence that graft quality and likelihood of a successful outcome rely on an accurate evaluation of the number of stem cells detected before transplantation as holoclones expressing high levels of the p63 transcription factor. No adverse effects related to the feeder layer have been observed and the regenerated epithelium was completely devoid of any 3T3-J2 contamination. CONCLUSION Cultures of limbal stem cells can be safely used to successfully treat massive destruction of the human cornea. We emphasize the importance of a discipline for defining the suitability and the quality of cultured epithelial grafts, which are relevant to the future clinical use of any cultured cell type.

In vitro transscleral iontophoresis of high molecular weight neutral compounds.

The aim of the work was to study in vitro, across porcine and human sclera, the effect of transscleral iontophoresis on the permeation of high molecular weight neutral dextrans, chosen as models of high molecular weight drugs. Iontophoretic (anodal and cathodal) and passive permeation experiments were performed through pig sclera using acetaminophen (MW=151Da) and labelled dextrans (MW between 4.4kDa and 120kDa) as neutral model compounds. Anodal iontophoresis of 120kDa dextran was also performed on human sclera. Membrane charge was also determined at different pH values. Both human and pig sclera show a net negative charge at pH 7.4 and in the presence of current an electroosmotic convective solvent flow in the anode-to-cathode direction takes place. During anodal iontophoresis, the electroosmotic flow (whose entity resulted approximately 4 microlcm(-2)h(-1)) was able to increase 2-6.5 times the transscleral flux of dextrans. Transscleral iontophoresis can be useful for enhancing the transport across the sclera of high molecular weight compounds, even though neutral. This result suggests a possible application of this technique for the non-invasive administration of new biotech drugs for the treatment of the posterior segment eye diseases.

Molecular and Clinical Characterization of Albinism in a Large Cohort of Italian Patients

PURPOSE The purpose of this study was to identify the molecular basis of albinism in a large cohort of Italian patients showing typical ocular landmarks of the disease and to provide a full characterization of the clinical ophthalmic manifestations. METHODS DNA samples from 45 patients with ocular manifestations of albinism were analyzed by direct sequencing analysis of five genes responsible for albinism: TYR, P, TYRP1, SLC45A2 (MATP), and OA1. All patients studied showed a variable degree of skin and hair hypopigmentation. Eighteen patients with distinct mutations in each gene associated with OCA were evaluated by detailed ophthalmic analysis, optical coherence tomography (OCT), and fundus autofluorescence. RESULTS Disease-causing mutations were identified in more than 95% of analyzed patients with OCA (28/45 [62.2%] cases with two or more mutations; 15/45 [33.3%] cases with one mutation). Thirty-five different mutant alleles were identified of which 15 were novel. Mutations in TYR were the most frequent (73.3%), whereas mutations in P occurred more rarely (13.3%) than previously reported. Novel mutations were also identified in rare loci such as TYRP1 and MATP. Mutations in the OA1 gene were not detected. Clinical assessment revealed that patients with iris and macular pigmentation had significantly higher visual acuity than did severe hypopigmented phenotypes. CONCLUSIONS TYR gene mutations represent a relevant cause of oculocutaneous albinism in Italy, whereas mutations in P present a lower frequency than that found in other populations. Clinical analysis revealed that the severity of the ocular manifestations depends on the degree of retinal pigmentation.

In-vitro permeation of bevacizumab through human sclera: effect of iontophoresis application

Objectives  Bevacizumab (Avastin) is a recombinant humanized monoclonal antibody used in ophthalmology (off‐label) for the treatment of neovascularization in diseases such as diabetic retinopathy and age‐related macular degeneration (wet form). Bevacizumab is currently administrated by repeated intravitreal injection, which can cause severe complications; a non‐invasive delivery route is therefore desirable. The passive permeation of bevacizumab through isolated human sclera was evaluated and the iontophoretic technique was explored as a method to enhance its transscleral transport in vitro.

Non-length-dependent small fibre neuropathy. Confocal microscopy study of the corneal innervation

Background It has been recently observed that small fibre neuropathy (SFN) may present as distal symmetrical polyneuropathy and with atypical non-length-dependent pattern. Objective To describe a small series of patients with non-length-dependent SFN, investigating corneal innervation with corneal confocal microscopy (CCM). Methods Evaluation of the corneal nerve fibre density using CCM in six women with non-length-dependent SFN. The patients were characterised by sensory disturbance involving proximal regions of the limbs, face and trunks, and the diagnosis was confirmed by the findings of decreased intraepidermal nerve fibre density on skin biopsy. Results Six women, aged 35–64, had non-length-dependent SFN, related to Crohn disease, impaired glucose tolerance and Sjögrens syndrome, or idiopathic (three cases). In all patients, CCM demonstrated decreased corneal nerve fibre density (12.5–23.4/mm2; normal, >30.6/mm2). Conclusion Non-length-dependent SFN may represent an intriguing diagnostic problem because of its puzzling presentation and the need for special investigations for its confirmation. In this perspective, CCM may provide a useful, non-invasive tool to complement the diagnostic workup.

Closed-chamber pulling-injection system for donor graft insertion in endothelial keratoplasty

I describe the closed-chamber pulling-injection (CCPI) technique to improve surgical control and safety of graft insertion into the anterior chamber during Descemet-stripping automated endothelial keratoplasty (DSAEK). In CCPI, the graft is rolled up (endothelium facing inward) in a closed transparent insertion device, allowing it to be drawn into a well-formed anterior chamber and unfolded smoothly. The technique has been used successfully to treat corneal decompensation in 72 consecutive cases. The results indicate that using the CCPI technique, both average and complicated cases can benefit from the numerous advantages provided by endothelial keratoplasty.

Multiple effects of adenosine in the arterially perfused mammalian eye. Possible mechanisms for the neuroprotective function of adenosine in the retina

It has been postulated that the major physiological role of adenosine is protection of the central nervous system in conditions such as ischemia, hypoxia, or prolonged neuronal excitation. Under these conditions adenosine is released, and exerts multiple effects, including vasodilation, inhibition of neuronal activity, and enhancement of glycogenolysis, resulting in neuroprotection. In this article, published as well as unpublished data on the multiple effects of exogenous adenosine and application of adenosine-related agents, performed using the arterially perfused cat eye, will be reviewed and discussed within the framework of the neuroprotective role of adenosine. The isolated, arterially perfused eye preparation has the advantage of combining integrity of the eye structure, exact control of arterial concentration and timing of applied pharmacological agents, and access to electrophysiological parameters of both retina and optic nerve, as well as the ability to control and monitor perfusate flow. The absence of red blood cells in the perfusate prevents adenosine from being metabolized prior to reaching the eye.

From discovery to approval of an advanced therapy medicinal product-containing stem cells, in the EU

In 1997, the human corneal epithelium was reconstructed in vitro and transplanted on patients. Later, it became a routine treatment, before regulations considered advanced therapy medicinal products and drugs on the same lines. Manufacturing, before and after good manufacturing practice setting, was established in different facilities and the clinical application in several hospitals. Advanced therapy medicinal products, including stem cells, are unique products with different challenges than other drugs: some uncertainties, in addition to benefit, cannot be avoided. This review will focus on all recent developments in the stem cell-based corneal therapy.

Dynamic imaging of accommodation by swept-source anterior segment optical coherence tomography

Purpose To study the accommodation process in normal eyes using a commercially available clinical system based on swept‐source anterior segment optical coherence tomography (AS‐OCT). Setting Ophthalmology Department, University of Parma, Italy. Design Evaluation of diagnostic technology. Methods Right eyes were analyzed using swept‐source AS‐OCT (Casia SS‐1000). The optical vergence of the internal coaxial fixation target was adjusted during imaging to obtain monocular accommodation stimuli with different amplitudes (0, 3.0, 6.0, and 9.0 diopters [D]). Overlapping of real and conjugate OCT images enabled imaging of all the anterior segment optical surfaces in a single frame. Central corneal thickness (CCT), anterior chamber depth (ACD), and lens thickness were extracted from the OCT scans acquired at different static accommodation stimulus amplitudes. The crystalline lens was analyzed dynamically during accommodation and disaccommodation by acquiring sequential OCT images of the anterior segment at a rate of 8 frames per second. The lens thickness was extracted from the temporal sequence of OCT images and plotted as a function of time. Results The study analyzed 14 eyes of 14 subjects aged 18 to 46 years. During accommodation, the decrease in the ACD was statistically significant (P < .05), as were the increase in the lens thickness (P < .001) and the slight movement forward of the lens central point (P < .01). The CCT and anterior chamber width measurements did not change statistically significantly during accommodation. The lens thickness at 0 D was positively correlated with age (P < .01). Conclusion High‐resolution real‐time imaging and biometry of the accommodating anterior segment can be effectively performed using a commercially available swept‐source AS‐OCT clinical device. Financial Disclosure No author has a financial or proprietary interest in any material or method mentioned.

Corneal thickness mapping by 3D swept-source anterior segment optical coherence tomography

Purpose:  To assess accuracy and repeatability of central corneal thickness (CCT) measurements obtained by swept‐source anterior segment optical coherence tomography (AS‐OCT), spectral‐domain retinal OCT with corneal module and ultrasound pachymetry (USP), and to assess repeatability of pachymetric mapping with AS‐OCT.