Investigating G-quadruplex structures in RPGR gene: Implications for understanding X-linked retinal degeneration.

Aims: This pilot study investigates the potential pathogenic role of G-quadruplex (G4) structures in RPGR-associated retinal degeneration, starting from a case of suspected X-linked form affected family. We hypothesize that the stabilization of these structures might alter DNA replication and transcription, inducing genetic instability and influencing gene expression. Main methods: We conducted whole genome amplification experiments and next-generation sequencing to detect the blockade of polymerase activity by G4 structures. Our specific focus was the RPGR gene, which hosts a high concentration of predicted G4-forming motifs and is implicated in most X-linked retinal degeneration cases. To understand the potential interference of G4 structures, we applied computational and 3D molecular modeling to visualize interferences in DNA replication and transcription regulation. Key findings: Our data confirmed the obstruction of DNA polymerase enzymes by G4 structures, particularly when stabilized by the compound pyridostatin. This obstruction was evident in the reduced amplification of RPGR gene regions and a shift in the start/end sites of putative G4 motifs. Moreover, the modeling indicated a potential disruption of critical promoter elements and RNA polymerase binding, which could drastically alter gene expression. Significance: Our findings suggest that G4 formation in the RPGR gene could lead to genetic instability and affect the expression of RPGR, contributing to retinal dystrophy. Moreover, this study underscores the broader implications of G4 structures in other genetic disorders. Improved understanding of G4 structures could reveal novel therapeutic targets to combat genetic disorders, promoting the advancement of personalized medicine and precision health.

Human retinal secretome: A cross-link between mesenchymal and retinal cells.

In recent years, mesenchymal stem cells (MSC) have been considered the most effective source for regenerative medicine, especially due to released soluble paracrine bioactive components and extracellular vesicles. These factors, collectively called the secretome, play crucial roles in immunomodulation and in improving survival and regeneration capabilities of injured tissue. Recently, there has been a growing interest in the secretome released by retinal cytotypes, especially retinal pigment epithelium and Müller glia cells. The latter trophic factors represent the key to preserving morphofunctional integrity of the retina, regulating biological pathways involved in survival, function and responding to injury. Furthermore, these factors can play a pivotal role in onset and progression of retinal diseases after damage of cell secretory function. In this review, we delineated the importance of cross-talk between MSCs and retinal cells, focusing on common/induced secreted factors, during experimental therapy for retinal diseases. The cross-link between the MSC and retinal cell secretomes suggests that the MSC secretome can modulate the retinal cell secretome and vice versa. For example, the MSC secretome can protect retinal cells from degeneration by reducing oxidative stress, autophagy and programmed cell death. Conversely, the retinal cell secretome can influence the MSC secretome by inducing changes in MSC gene expression and phenotype.

Effect of COVID-19 lockdown on refractive errors in Italian children aged 5-12 years: A multi-center retrospective study.

PURPOSE: to explore the potential consequences of the COVID-19 lockdown on the prevalence of myopia among Italian children aged 5-12 years. MATERIALS AND METHODS: retrospective multicenter study conducted in Italy. Population: children aged 5-12. Selection: random selection of children who received an eye exam between 2016 to 2021. Inclusion criteria: healthy children presenting for a routine eye exam. Exclusion criteria: presence of ocular comorbidities other than refractive error, such as blepharoptosis, media opacities, corneal or retinal dystrophies, strabismus, amblyopia, or concurrent therapy with atropine 0.01%. Outcome measure: age and spherical equivalent (SE) measured in diopters (D) in the right eye (RE) in cycloplegia. Statistical analysis: ANOVA test. RESULTS: total of 803 children. In the years prior to COVID-19, the mean SE ± SD of healthy age-school children was: 0.54 ± 1.49 D in 2016; 0.43 ± 1.84 D in 2017; 0.34 ± 1.41 D in 2018; 0.35 ± 1.75 D in 2019 (ANOVA, p = .659). In 2021, the mean SE changed to -0.08 ± 1.44 D (ANOVA, p = .005). Mean age was comparable in all groups (ANOVA, p = .307). The prevalence of myopes (SE ≤-0.5D) and hyperopes (SE ≥ 2D) was respectively 24.10% and 9.64% among children aged 60-96 months, and 63.86% and 6.02% among children aged 97-144 months. These values represent a statistically-significant increase in the number of myopes (Chi-square, p = .016) and decrease in the number of hyperopes (Chi-square, p = .001), as compared to previous years (.06 and.48 respectively). CONCLUSION: this retrospective study shows a statistically-significant decrease in the mean SE in children aged 5-12 in the year following the COVID-19 lockdown. The percentage of myopes has increased significantly, while the percentage of hyperopes has decreased. The lifestyle changes caused by the lockdown led children to spend more time on near-work activities and digital devices, and less time outdoors. These are known risk factors for the development and progression of myopia. Studies in different countries are encouraged.

Epitranscriptome Analysis of Oxidative Stressed Retinal Epithelial Cells Depicted a Possible RNA Editing Landscape of Retinal Degeneration.

Oxidative stress represents one of the principal causes of inherited retinal dystrophies, with many related molecular mechanisms still unknown. We investigated the posttranscriptional RNA editing landscape of human retinal pigment epithelium cells (RPE) exposed to the oxidant agent N-retinylidene-N-retinyl ethanolamine (A2E) for 1 h, 2 h, 3 h and 6 h. Using a transcriptomic approach, refined with a specific multialgorithm pipeline, 62,880 already annotated and de novo RNA editing sites within about 3000 genes were identified among all samples. Approximately 19% of these RNA editing sites were found within 3' UTR, including sites common to all time points that were predicted to change the binding capacity of 359 miRNAs towards 9654 target genes. A2E exposure also determined significant gene expression differences in deaminase family ADAR, APOBEC and ADAT members, involved in canonical and tRNA editing events. On GO and KEGG enrichment analyses, genes that showed different RNA editing levels are mainly involved in pathways strongly linked to a possible neovascularization of retinal tissue, with induced apoptosis mediated by the ECM and surface protein altered signaling. Collectively, this work demonstrated dynamic RNA editome profiles in RPE cells for the first time and shed more light on new mechanisms at the basis of retinal degeneration.

Effects of Photo-Biomodulation in Stargardt Disease.

PURPOSE: Stargardt disease (STGD1) is the most common hereditary macular degeneration and currently no treatments have been approved to interrupt the progression of the disease. The aim of this study was to evaluate the efficacy of photo-biomodulation in patients with Stargardt disease 1 year after treatment, considering as primary outcomes improvements in best corrected visual acuity (BCVA), microperimetry, and pattern electroretinography (PERG) amplitude. MATERIALS AND METHODS: Ninety eyes of 45 patients with a diagnosis of Stargardt disease stage 1 were prospectively recruited at the University of Bologna. Selected patients were treated with photo-biomodulation therapy for 1 year for 10 minutes a day, twice a day, and 5 days per week for 12 months. A light emitting diode (LED) of 10 Hz and wavelength 650 nm was used. BCVA and microperimetry were assessed before treatment and 1 year after photo-biomodulation, whereas PERG was assessed at baseline, 1, 3, 6, and 12 months after treatment. RESULTS: BCVA improved from 0.7 [interquartile range (IQR)=0.6-0.9)] to 0.4 (IQR=0.2-0.5)] after treatment (95% CI=-0.3, -0.35; p=<0.001) and PERG negative wave at 35 ms (N35)-positive wave at 50 ms (P50) amplitude improved from 0.9 (IQR=0.74-1.21) to 2.12 (95% CI=1.11, 1.28; p<0.001). MP-1 Microperimetry improved from 26.5% (IQR=16.75-40) of median preferred retinal loci (PRL) within an area of 2° of diameter to 38% (IQR=29-50.25; 95% CI=10.50, 14.50; p=<0.001); on the other hand, PRL within 4° of diameter raised from 72% (IQR=51.5-80) to 80% (IQR=76-87) after photo-biomodulation (95% CI=4.50, 7.50; p<0.001). Also, patients with an improvement of more than 1.205 PERG N35-P50 ratio amplitude at 3 months and more than 1.29 at 6 months tended to present an improvement of more than 0.2 logMar in BCVA at 12 months (p=0.28, AUC=0.630; p=0.30, AUC=0.636). CONCLUSION: BCVA, PERG, and MP-1 significantly improved 1 year after treatment. Also, improvement of PERG N35-P50 amplitude at 3 and 6 months was correlated to BCVA improvement at 1 year, suggesting that PERG could be useful in predicting visual outcome after photo-biomodulation.

Risk factors in central retinal vein occlusion: A multi-center case-control study conducted on the Italian population : Demographic, environmental, systemic, and ocular factors that increase the risk for major thrombotic events in the retinal venous system.

PURPOSE: To explore the risk factors for central retinal vein occlusion (CRVO) by comparing a large sample of patients with healthy controls. MATERIALS AND METHODS: Multi-center case-control study. The study group includes patients affected by central retinal vein occlusion, confirmed angiographically, aged 50 years old or above (Group A). The control group includes healthy subjects without an history of retinal vein occlusion (Group B). Outcome measures: age, gender, active smoking, presence of uncontrolled arterial hypertension (uHTN), presence of the following comorbidities: diabetes mellitus type II (DMII), chronic liver disease (CLD), chronic kidney disease (CKD), thyroid disease (TD), systemic lupus erythematosus (SLE), hyperhomocystenemia (HHcy), dyslipidemia (DLip), carotid artery disease (CAD), glaucoma, atrial fibrillation (AF), migraine headache (MH), chronic obstructive pulmonary disease (COPD), obstructive sleep apnea syndrome (OSAS), history of myocardial infarction (MI). Odds-ratios were calculated with logistic regression analysis. RESULTS: A total of 203 patients (Group A) and 339 controls (Group B). Statistically-significant differences were found for the following variables: age (OR: 1.109 [1.081-1.138], p < .001), active smoking (OR: 2.048 [1.210- 3.466], p < .008), DMII (OR: 4.533 [2.097-9.803], p < .001), HHcy (OR: 4.507 [2.477-10.001 ], p < .001), DLip (OR: 2.255 [1.352-3.762], p = .002), CAD (OR: 6.632 [2.944- 14.942], p < .001), glaucoma (OR: 4.656 [2.031-10.673], < .001), OSAS (OR: 1.744 [1.023-2.975], < .041), uHTN (OR: 3.656 [2.247-5.949], < .001). No statistically-significant differences were found for the other variables. CONCLUSIONS: Older age, active smoking, as well as presence of DMII, HHcy, DLip, CAD, glaucoma, OSAS, and uHTN, all increase the risk for CRVO. A comprehensive assessment of patients with CRVO is paramount. Adequate control of all the aforementioned risk factors is likely of great significance in reducing the incidence of CRVO among the general population, and it likely plays an important role in improving the prognosis following the occlusive event.

The effect of diabetic retinopathy on standing posture during optic flow stimulation.

BACKGROUND: Diabetic retinopathy is a principal cause of visual damage and blindness, in which laser treatment offers proven therapy. The progressive degeneration of the retina, secondary to diabetes, is believed to cause postural instability although this is not well documented. The aim of this research was to assess how optic flow stimuli contribute to the control of stance in people with impaired retinal functions. RESEARCH QUESTION: Does the different retinal functionality correspond to different specific patterns of movements and muscles recruitment? METHODS: Postural mechanisms and motor strategies were measured by testing subjects in quiet stance on a force platform with surface electromyography under different optic flow stimulations. Root mean square values of the center of pressure time-varying signals and normalized EMG values were used to evaluate the postural sway. RESULTS: People with diabetic retinopathy, and to a greater extent laser group, were more unstable than healthy subjects. The greater amplitude of the body sway observed in the retinopathy group, and especially in the laser group, could be an expression of the difficulty for this population in processing this kind of visual information. SIGNIFICANCE: The increase in muscle activity indicates that there are musculoskeletal and postural changes in the lower limb musculature with increasing severity of diabetic retinopathy. An impaired retinal function might negatively affect postural control in a way that is dependent on the severity of retinal damage.

Prolonged face mask use might worsen dry eye symptoms.

Purpose: The aim of this study was to explore whether prolonged and consistent face mask use might be associated with worsening of dry eye symptoms in patients with dry eye disease (DED). Methods: Subjects with a previous diagnosis of DED. Their OSDI scores were compared with those recorded in Fall 2019 using the Wilcoxon paired test. Participants were stratified by face mask use: heavy (Group A) or standard (Group B) face mask users. Heavy use was defined as wearing any type of face mask for at least 6 hours a day, at least 5 days per week in the last 2 months. Results: 67 subjects (mean age: 45.27 ± 10.06 SD years, 40% males and 60% females). Median OSDI score in Fall 2019: 18.75. Median OSDI in Spring 2020: 20.83. The Hodges-Lehmann median difference was 2.09 (95% CI [1.05, 4.17]) (P < 0.0001). The population was then stratified into heavy and standard face mask users: Group A included heavy users (31 subjects; mean age: 42.81 ± 10.48 SD years; 35% males and 65% females), Group B included standard users (36 subjects; mean age: 47.39 ± 9.31 SD years; 44% males and 56% females). The Hodges-Lehmann median difference was 5.21 (95% CI [3.13, 7.29]) in Group A (P < 0.0001), and 1.04 (95% [0],[ 2.08]) in Group B (P = 0.0177). Conclusion: Prolonged and consistent face mask use is associated with an increase in OSDI scores. Whether face mask use is responsible for the worsening of symptoms of DED remains to be elucidated.

Efficacy and safety of intravitreal Fluocinolone Acetonide microimplant (ILUVIEN®) in patients with chronic diabetic macular edema: 1 year follow-up.

PURPOSE: Evaluate the efficacy and safety of intravitreal 0.19 mg fluocinolone acetonide (FAc) micro implant in patients with chronic diabetic macular edema (cDME). METHODS: Prospective study recruiting subjects with cDME. Inclusion criteria: cDME for at least 2 years documented with OCT imaging; pseudophakia; previous treatments with laser photocoagulation and intravitreal injections of anti-VEGF and/or dexamethasone. Exclusion criteria: phakia; ocular hypertension; tractional component visible on OCT; glaucoma; previous vitrectomy. Outcome measures included best-corrected visual acuity (BVCA), intraocular pressure (IOP), and central macular thickness (CMT), measured 1, 3, 6, and 12 months post-injection. Data were compared with the Friedman test and significance was set at p < 0.05. RESULTS: A total of 18 eyes with a median duration of cDME of 45 months (25-118 months). The 77% of subjects either maintained or improved their BVCA. About 17% and 33% of subjects showed an improvement of 15 ETDRS letters or more at 3 and 12 months respectively. The 17% and 28% of subjects showed a CMT <250 microns at 3 and 12 months, respectively. The median change in CMT thickness was of -370 and -373.5 microns at 3 and 12 months post-injection respectively (p-value is 0.025). Changes in median IOP at 3 and 12 months post-injection were not statistically significant (p-value is 0.210). Ocular hypertension (OHT) was detected in two eyes (11%). CONCLUSION: The FAc micro implant has proved efficacy in improving and/or maintaining BVCA in 77% of patients with cDME up to 12 months post-injection. Ocular hypertension is the most common side effect but responds well to topical therapy.

Expression of Pro-Angiogenic Markers Is Enhanced by Blue Light in Human RPE Cells.

Inherited retinal dystrophies are characterized by photoreceptor death. Oxidative stress usually occurs, increasing vision loss, and oxidative damage is often reported in retinitis pigmentosa (RP). More than 300 genes have been reported as RP causing. In contrast, choroidal neovascularization (CNV) only occasionally develops in the late stages of RP. We herein study the regulation of RP causative genes that are likely linked to CNV onset under oxidative conditions. We studied how the endogenous adduct N-retinylidene-N-retinylethanolamine (A2E) affects the expression of angiogenic markers in human retinal pigment epithelium (H-RPE) cells and a possible correlation with RP-causing genes. H-RPE cells were exposed to A2E and blue light for 3 and 6h. By transcriptome analysis, genes differentially expressed between A2E-treated cells and untreated ones were detected. The quantification of differential gene expression was performed by the Limma R package. Enrichment pathway analysis by the FunRich tool and gene prioritization by ToppGene allowed us to identify dysregulated genes involved in angiogenesis and linked to RP development. Two RP causative genes, AHR and ROM1, can be associated with an increased risk of CNV development. Genetic analysis of RP patients affected by CNV will confirm this hypothesis.

Conjunctivitis can be the only presenting sign and symptom of COVID-19.

Five cases of non-remitting conjunctivitis turned out to be the sole presenting sign and symptom of COVID-19. These patients tested positive on RT-PCR of naso-pharyngeal swabs and developed no fever, malaise, or respiratory symptoms throughout the course of their illness. They all fully recovered. In the current efforts to fight the spread of this virus, authors want to emphasize that atypical clinical presentations of COVID-19 can occur and a high level of suspicion should be maintained. Ocular involvement and transmission of SARS-CoV-2 should never be overlooked. In fact, conjunctival mucosae are susceptible to respiratory viruses and remain an important point of entry. For this reason, eye protection in the form of goggles or a face shield should be considered essential for all healthcare providers, even when taking care of patients who are not showing typical symptoms of COVID-19.

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.

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 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.

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.

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)-Rpe(rd12)/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.

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.

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 microg/10 microl 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.

A novel stem cell tag-less sorting method.

Growing interest in stem cell research has led to the development of a number of new methods for isolation. The lack of homogeneity in stem cell preparation blurs standardization, which however is recommended for successful applications. Among stem cells, mesenchymal stem cells (MSCs) are promising candidates for cell therapy applications. This paper presents a fractionation protocol based on a tag-less, flow-assisted method of purifying, distinguishing and sorting MSCs. The protocol entails a suspension of cells in a transport fluid being injected into a ribbon-like capillary device by continuous flow. In a relatively short time (about 30 min) sorted cells are collected. The protocol has been applied to the improvement of MSC isolation, with a specific view to reducing cell manipulation operations, keeping instrumental simplicity and increasing analytical information for cell characterization. Applications such as MSC purification from epithelial contaminants, MSC characterization from various human sources and sorting of MSC subpopulations with high differentiation potential are described. The low cost, full biocompatibility and scale-up potential of the protocol presented could make the procedure attractive for stem cell selection.