Sergio Zaccaria Scalinci

A simple method for identifying bone marrow mesenchymal stromal cells with a high immunosuppressive potential

BACKGROUND AIMS The beneficial activity of mesenchymal stromal cells (MSC) in allogeneic hematopietic stem cell transplantation requires correct use in terms of cell dose and timing of infusion and the identification of biomarkers for selection. The immunosuppressive bone marrow (BM)-derived MSC (BM-MSC) functions have been associated with the production of soluble HLA-G molecules (sHLA-G) via interleukin (IL)-10. We have established a reliable method for evaluating BM-MSC HLA-G expression without the influence of peripheral blood mononuclear cells (PBMC). METHODS Thirteen BM-MSC from donors were activated with recombinant IL-10 or co-cultured with 10 different phytohemagglutinin (PHA)-treated PBMC (PHA-PBMC). Membrane-bound and sHLA-G expression was evaluated by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively; lymphoproliferation was measured by (methyl-(3)H)thymidine. RESULTS The results demonstrated the ability of IL-10 to stimulate both membrane-bound and sHLA-G production by BM-MSC. The levels of HLA-G expression induced by IL-10 in BM-MSC were associated with the inhibition of PHA-PBMC proliferation (sHLA-G, P = 0.0008, r = 0.9308; membrane HLA-G, P = 0.0005, r = 0.9502). CONCLUSIONS We propose the evaluation of sHLA-G production in IL-10-treated BM-MSC cultures as a possible marker of immunoregulatory function.

Potential role of intravitreal human placental stem cell implants in inhibiting progression of diabetic retinopathy in type 2 diabetes: neuroprotective growth factors in the vitreous

Background: Intravitreal injection of human mesenchymal stem cells has been shown to be effective in slowing the progression of diabetic retinopathy in an animal model of chemically induced diabetes mellitus. We studied changes in growth factor levels released from human mesenchymal stem cells in the vitreous cavity as well as changes in growth factor levels in host retinal neurons following intravitreal injection. Methods: Twenty-two Lewis rats were treated with an intravitreal human mesenchymal stem cell microinjection. Determination of neurotrophic factors released by human mesenchymal stem cells in the vitreous was carried out using real-time polymerase chain reaction. Results: Detectable levels of neurotrophic factors were identified postoperatively in the vitreous of all rats. Conclusion: Increased intravitreal and retinal concentrations of neuroprotective growth factors in rats confirm the neuroprotective activity of human mesenchymal stem cells in diabetic retinopathy.

Cell surgery and growth factors in dry age-related macular degeneration: visual prognosis and morphological study

Background The aim of this research was to study the overall restoration effect on residual retinal cells through surgically grafted autologous cells onto the surrounding tissue, choroid and retina in order to produce a constant secretion of growth factors (GFs) in dry age-related macular degeneration (AMD) patients. Results 6 months after surgery, several values were statistically significant in the group with higher RTA. Also patient compliance analysis (PCA) in relation to functional change perception appeared to be very good. Methods Thirty-six eyes of 25 patients (range 64-84 years of age) affected by dry AMD were included in study, and divided in two groups by spectral domain-optical coherence tomography (SD-OCT): group A with retinal thickness average (RTA) less than 250 microns (μm) and group B with RTA equal to or more than 250 μm. Adipocytes, adipose-derived stem cells from the stromal-vascular fraction, and platelets from platelet-rich plasma were implanted in the suprachoroidal space. Particularly, the following parameters were evaluated: best corrected visual acuity (BCVA) for far and near distance, retinal thickness maps, scotopic and photopic electroretinogram (ERG), and microperimetry (MY). All statistical analyses were performed with STATA 14.0 (Collage Station, Texas, USA). Conclusions The available set of GFs allowed biological retinal neuroenhancement. After 6 months it improved visual performance (VP), but the increase was better if RTA recorded by OCT was higher, probably in relation to the presence of areas with greater cellularity.

Effect of basic fibroblast growth factor and cytochrome c peroxidase combination in transgenic mice corneal epithelial healing process after excimer laser photoablation

Purpose: To evaluate the role of prepared basic fibroblast growth factor (bFGF) and cytochrome c peroxidase (CCP) combination eyedrops in corneal epithelial healing of transgenic mice (B6(A)-Rperd12/J) after excimer laser photoablation. Materials and methods: In this prospective study, 216 eyes of 108 mice underwent bilateral photorefractive keratectomy. We considered 4 groups: A, B, C, and D. Group A received standard topical postoperative therapy with tobramycin, diclofenac, and dexamethasone eyedrops plus CCP at 3 drops per day for a week or until corneal re-epithelialization was achieved. Group B received standard topical postoperative therapy plus bFGF eyedrops and phosphate-buffered saline (PBS) 3 drops per day for a week or until corneal re-epithelialization was complete. In group C, 1 eye received standard topical postoperative therapy plus CCP eyedrops, bFGF eyedrops, and PBS 3 drops per day for a week or until corneal re-epithelialization was complete. Control eyes (group D) received a standard topical postoperative therapy plus placebo eyedrops. Mice were followed-up for a week from the day after the surgery to evaluate the rate of corneal re-epithelialization. Results: Data were analyzed by ANOVA using the XLSTAT 2010 software. Eyes in group A, B, and C healed completely before the fifth postoperative day, achieving, respectively, a re-epithelialization time of 92 hours ± 10 SD, 90 hours ± 12 SD, and 86 hours ± 12 SD. Group D had a re-epithelialization time of 121 hours ± 8 SD (P < 0.05). No side effects or toxic effects were documented. Conclusions: Results suggest that re-epithelialization after phototherapeutic keratectomy can benefit from topical therapy with CCP/bFGF combination eyedrops. Further clinical studies are needed to evaluate the long-term effectiveness of these eyedrops to prevent corneal haze.

Effect of Basic Fibroblast Growth Factor in Transgenic Mice: Corneal Epithelial Healing Process after Excimer Laser Photoablation

Purpose: To evaluate the role of preparedbasic fibroblast growth factor (bFGF) eyedrops in corneal epithelial healing of transgenic mice after excimer laser photoablation. Materials and Methods: In our prospective case series, 60 eyes of 30 mice had uneventful bilateral excimer laser photoablation. In each mouse, 1 eye received the standard topical postoperative therapy with tobramicin, diclofenac and dexamethasone eyedrops, plusbFGF eyedrops 5 μg/10 μl PBS 3 times a day for 1 week, or until corneal reepithelialization was complete. The fellow eye served as the control and received the standard postoperative therapy plus placebo. The mice were monitored daily, commencing the day after surgery and for 7 days, in order to evaluate the corneal reepithelialization rate by using a video slitlamp camera with a cobalt blue light. The mean diameter of the corneal wounds was measured. Videotaped images were recorded and analyzed by computer planimetry. Results: All the eyes treated withbFGF eyedrops healed completely before day 5 after surgery, with a mean reepithelialization time of 90 ± 12 h (standard deviation); the mean reepithelialization time was 124 ± 10 h in those eyes receiving placebo. There were no statistically significant differences between the 2 groups in corneal haze presentation during the follow-up, perhaps because the time period was too brief (7 days). However, corneal clarity on slitlamp biomicroscopy was greater in the study group than in the control group. No side effects or toxic effects were documented. Conclusions: These data suggest that the bFGF significantly accelerates epithelial healing after excimer photoablation. A further clinical study should be performed to prove the results obtained in this study as well as the long-term efficacy of bFGF to prevent corneal haze.

Effect of the combination of basic fibroblast growth factor and cysteine on corneal epithelial healing after photorefractive keratectomy in patients affected by myopia

BACKGROUND This study sought to evaluate the effect of basic fibroblast growth factor eye drops and cysteine oral supplements on corneal healing in patients treated with photorefractive keratectomy (PRK). MATERIALS AND METHODS One hundred and twenty patients treated bilaterally with PRK for myopia were enrolled at one of two eye centers (Clinica Santa Lucia, Bologna, Italy and Department of Ophthalmology, University of Magna Graecia, Catanzaro, Italy) and were treated at the former center. Sixty patients included in the study group (Group 1) were treated postoperatively with topical basic fibroblast growth factor plus oral L-cysteine supplements, whereas 60 subjects included in the control group (Group 2) received basic fibroblast growth factor eye drops. We recorded the rate of corneal re-epithelialization and patients were followed-up every 30 days for 6 months. Statistical analyses were performed on the collected data. RESULTS The eyes in Group 1 demonstrated complete re-epithelialization at Day 5, whereas the eyes in Group 2 achieved this status on Day 6. No side-effects were reported. CONCLUSIONS Patients treated with basic fibroblast growth factor eye drops and L-cysteine oral supplements benefit from more rapid corneal re-epithelialization. In human eyes, this combination treatment appeared to be safe and effective in accelerating corneal surfacing after surgery. FINANCIAL DISCLOSURE No author has any financial or proprietary interest in any material or method used in this study. TRIAL REGISTRATION Current Controlled Trials ISRCTN73824458.

Effect of Cysteine in Transgenic Mice on Healing of Corneal Epithelium after Excimer Laser Photoablation

Purpose: To evaluate the role of prepared cysteine per os in corneal epithelial healing in transgenic mice (B6(A)-Rpe65rd12/J) after excimer laser photoablation. Materials and Methods: In our prospective case series, 60 eyes of 30 mice had uneventful bilateral excimer laser photoablation. A first group, composed of 15 mice, received standard topical post-operative therapy with tobramycin, diclofenac and dexamethasone eyedrops, pluscysteine 5 mg 5 μg/10 μl phosphate-buffered saline (3 times a day for 1 week) per os or until corneal re-epithelialization was complete. The control group of 15 mice received standard post-operative therapy plus placebos. The mice were monitored daily, commencing the day after surgery, for 7 days to evaluate the corneal re-epithelialization rate using a video slit-lamp camera with a cobalt blue light. The mean diameters of corneal wounds were measured. Videotaped images were recorded and analysed by computer planimetry. Results: All the eyes of the mice treated withcysteine healed completely by day 5 after surgery, with a mean re-epithelialization time of 92 ± 10 h (SD); the mean re-epithelialization time was 125 ± 8 h in eyes that received the placebo. There were no statistically significant differences between the 2 groups in corneal haze presentation during follow-up, perhaps because the time period was too brief (7 days). However, corneal clarity in the study group was greater than in the control group, as shown on slit-lamp biomicroscopy. No side effects or toxic effects were documented. Conclusions: These data suggest that cysteine significantly accelerates epithelial healing after excimer photoablation. A further clinical study should be performed to confirm the results obtained in this study, and the long-term efficacy of cysteine in preventing corneal haze.

Effect of Cytochrome c Peroxidase on the Corneal Epithelial Healing Process after Excimer Laser Photo-Ablation in Transgenic Mice

Aim: To evaluate the role of commercially prepared cytochrome c peroxidase eye drops in corneal epithelial healing of transgenic B6(A)-Rpe65rd12/J mice after excimer laser photo-ablation. Materials and Methods: In our prospective animal series, 72 eyes of 36 mice had uneventful bilateral excimer laser photo-ablation. In each mouse, one eye received standard topical postoperative therapy with tobramycin, diclofenac, and dexamethasone eye drops plus cytochrome c peroxidase eye drops (two drops three times a day for 1 week or until corneal re-epithelialization was complete, corresponding to 15,000 IU/day). The fellow eye served as the control and received standard postoperative therapy plus placebo. The mice were monitored daily, commencing on the day after surgery, for 7 days to evaluate the corneal re-epithelialization rate using a video slit lamp camera with cobalt blue light. The mean diameter of the corneal wounds was measured. Videotaped images were recorded and analyzed by computer planimetry. Results: All eyes treated with cytochrome c peroxidase eye drops healed completely before day 5 after surgery, with a mean re-epithelialization time of 92 ± (SD) 10 h; the mean re-epithelialization time was 121 ± 8 h in the eyes receiving placebo (p < 0.05). There were no statistically significant differences between the two groups in corneal haze presentation during the follow-up period (p = 0.70), perhaps because the observation period was too short (7 days). However, the corneal clarity, on slit lamp biomicroscopy, in the study group was higher than that in the control group. No side effects or toxic effects were observed. Conclusions: These data suggest that cytochrome c peroxidase significantly accelerates epithelial healing after phototherapeutic keratectomy. Further clinical studies should be performed to prove the results obtained in this study and the long-term efficacy of cytochrome c peroxidase to prevent corneal haze.

Regenerative Therapy by Suprachoroidal Cell Autograft in Dry Age-related Macular Degeneration: Preliminary In Vivo Report

This study is aimed at examining whether a suprachoroidal graft of autologous cells can improve best corrected visual acuity (BCVA) and responses to microperimetry (MY) in eyes affected by dry Age-related Macular Degeneration (AMD) over time through the production and secretion of growth factors (GFs) on surrounding tissue. Patients were randomly assigned to each study group. All patients were diagnosed with dry AMD and with BCVA equal to or greater than 1 logarithm of the minimum angle of resolution (logMAR). A suprachoroidal autologous graft by Limoli Retinal Restoration Technique (LRRT) was carried out on group A, which included 11 eyes from 11 patients. The technique was performed by implanting adipocytes, adipose-derived stem cells obtained from the stromal vascular fraction, and platelets from platelet-rich plasma in the suprachoroidal space. Conversely, group B, including 14 eyes of 14 patients, was used as a control group. For each patient, diagnosis was verified by confocal scanning laser ophthalmoscope and spectral domain-optical coherence tomography (SD-OCT). In group A, BCVA improved by 0.581 to 0.504 at 90 days and to 0.376 logMAR at 180 days (+32.20%) postoperatively. Furthermore, MY test increased by 11.44 dB to 12.59 dB at 180 days. The different cell types grafted behind the choroid were able to ensure constant GF secretion in the choroidal flow. Consequently, the results indicate that visual acuity (VA) in the grafted group can increase more than in the control group after six months.

Comparative Study of the Effects of Clobetasone Butyrate and Indomethacin Ophthalmic Solutions after Radial Keratotomy or Epikeratophakia

SummaryIn each of 12 patients subjected to bilateral radial keratotomy, 1 eye was treated with indomethacin 0.5% ophthalmic solution and the other eye was treated with clobetasone butyrate 0.1% ophthalmic solution for a period of 60 days after surgery. The following assessments were carried out: (a) degree of inflammation; (b) subjective symptoms; (c) tonometric assessment; and (d) culture of lacrimal fluid before surgery and after 2 months of observation. No statistically significant differences in the postoperative regression of the correction were observed; however, inflammatory reactions were less evident with clobetasone, especially in patients with hypersensitivity conjunctivitis. An evaluation of clobetasone was then made in patients who underwent epikeratophakia. Clobetasone proved to be effective in the treatment of inflammatory reactions, without interfering in the course of re-epithelisation of the implanted lenticule.