Joseph Frucht-Pery

Nerve growth factor is preformed in and activates human peripheral blood eosinophils

BACKGROUND Recent studies have shown that nerve growth factor (NGF) is produced by and can act on several immune-inflammatory cells. OBJECTIVES The objective of this study was to study the effects of NGF on human peripheral blood eosinophils and assess whether these cells produce and store NGF. METHODS Eosinophils were purified by negative immunoselection (magnetic cell sorting systems, purity 98% to 100%) from 13 subjects (9 to 26 years old) with mild blood eosinophilia, mainly of allergic origin. Eosinophils were incubated with NGF (50 to 1000 ng/mL), and supernatants were collected for measurement of eosinophil peroxidase (EPO, 20 minutes, colorimetric enzymatic assay) and IL-6 (12 hours, ELISA). Eosinophil viability was evaluated by Trypan blue test (days 2, 3, and 4). NGF content in freshly isolated eosinophils, after ultrasound disruption, was determined with a 2-site immunoenzymatic assay. The presence of mRNA for NGF was evaluated by reverse transcription PCR. RESULTS NGF caused EPO release (highly significant at 1000 ng/mL NGF). IL-6 release from eosinophils was not higher than IL-6 spontaneously released into culture medium alone. NGF did not significantly affect the number of viable eosinophils. NGF was found in the eosinophil sonicates (1.5 to 17.8 pg/mL per 106 cells). Similarly, mRNA for NGF was detected by reverse transcription PCR in the freshly isolated eosinophils. CONCLUSIONS NGF activates human peripheral blood eosinophils from subjects with mild eosinophilia to selectively release inflammatory mediators. Eosinophils store and produce NGF. Therefore the capability of NGF to induce a secretory response and its production and storage by circulating human eosinophils suggest a possible role for NGF in conditions associated with eosinophilia, including allergic disease.

Intraoperative Application of Topical Mitomycin C for Pterygium Surgery

BACKGROUND Postoperative recurrence of pterygium occurs in many patients. The authors studied the recurrence rate of pterygium after administration of a single intraoperative dosage of topcial mitomycin C at the completion of pterygium excision. METHODS AND PATIENTS Eighty-one patients underwent excision of the pterygium, leaving the sclera bare. The first 60 patients were randomized into two treatment groups of 30 patients each. Their remaining 21 patients were offered mitomycin C. Group 1 included 49 patients (30 randomized and 19 of the remaining 21 patients) who received an intraoperative application of 0.02% (0.2 mg/ml) mitomycin C for 5 minutes, and group 2 included 32 patients (30 randomized and 2 of the remaining 21 patients) who received NaCl 0.9% instead of mitomycin C. Patients were followed from 12 to 28 months in a masked manner. RESULTS The pterygium recurred in 2 (4%) of 49 patients in group 1 and in 15 (46.7%) of the 32 patients in group 2 (P = 0.0001). A delay of epithelialization for 5 and 10 weeks occurred in two patients in group 2 and granuloma manifested in one patient in group 1. CONCLUSION This study indicates that intraoperative administration of a single dosage of 0.02% mitomycin C is an effective treatment for prevention of recurrence of pterygium.

The Use of Low-dose Mitomycin C for Prevention of Recurrent Pterygium

BACKGROUND The administration of high doses of topical mitomycin C after pterygium excision causes a variety of complications. METHODS Seventy-five patients who had advanced or recurrent pterygia underwent excision of pterygia, leaving the sclera bare. Patients were randomized in a masked fashion into three groups of 25 patients each. Patients in group 1 received topical 0.01% mitomycin C for 5 days, patients in group 2 received 0.02% mitomycin C for 5 days, and those in group 3 were treated with 1200 rad beta-irradiation. Patients were followed by a surgeon who was masked to the patient treatment. The mean follow-up period was 15.3 months. RESULTS The recurrence rate after pterygium surgeries was 8% in group 1, 4% in group 2, and 20% in group 3. There were no statistical differences between the study groups. Delay of epithelialization for 8 weeks in one patient and degenerative calcification of conjunctiva 18 months after surgery in another patient, both from group 2, were the only complications in this study. CONCLUSION This study indicates the advantage of 0.01% mitomycin C for post-operative prevention of recurrence of pterygium.

Treatment of conjunctival intraepithelial neoplasia with topical drops of mitomycin C.

Purpose. To evaluate the efficacy and risks of complications of topical mitomycin C (MMC) for small-size conjunctival intraepithelial neoplasia (CIN). Methods. Eight patients with clinically diagnosed CIN <8 mm were included in the study. Patients received topical drops of MMC, 0.02–0.04%, 4 times daily for 14 days. Retreatment was done when lesions were not eliminated or recurred after the first treatment. Results. Three patients remained disease free after one course of MMC application. Retreatment was done in four patients because of lesions that decreased in size but were not eliminated, and for regrowth in one case. After retreatment, the lesions were eradicated in four patients, whereas in one patient, the treatment failed, and the lesion was surgically excised. The complications of MMC use included mild conjunctival hyperemia in two patients and mild allergy in one patient, which resolved after discontinuation of the treatment. Conclusion. Application of topical MMC is an effective treatment for most but not all cases of small-size CIN.

Proliferative activity and p53 expression in primary and recurrent pterygia

PURPOSE To assess p53 expression and proliferative activity in primary and recurrent pterygia from the same eyes. DESIGN Retrospective comparative human tissue study. PARTICIPANTS Tissue from excised primary pterygia that did not recur (group A, n = 10) was compared with tissue from primary pterygia that recurred (group B, n = 10) and to the recurrent pterygia tissue that was excised from subjects in group B (group C, n = 10). Ten normal conjunctivas served as controls (group D). METHODS Sections from each pterygium were immunostained with the MIB-1 and bp53. 12 monoclonal antibodies that react with Ki-67 and p53 antigens, respectively. MAIN OUTCOME MEASURES Proliferative activity was calculated as the mean of the MIB-1 positive cell count per eyepiece grid in high magnification (x40) (positive cell count/grid). Percentage of positive cells of all cells in the grid area was evaluated in the p53-stained sections. RESULTS Proliferative activity was found in the epithelium overlying the pterygia and normal conjunctiva. The mean MIB-1 positive cell count/grid +/- standard error was 2.84 +/- 1.07, 1.74 +/- 0.82, 3.83 +/- 1.35, and 0.86 +/- 0.33 in groups A, B, C, and D, respectively (P = 0.17, Kruskal-Wallis). P53 staining was found in 50% of pterygia in groups A, B, and C; none of the normal conjunctival tissues showed p53 immunoreactivity. Four of five p53-positive tissues in group B were p53-negative in group C. In the p53-positive pterygia, less than 10% of cells were p53 positive. However, p53-positive pterygia had higher mean MIB-1 positive cell count/grid +/- standard error as compared with the p53-negative lesions, 4.56 +/- 0.94 vs 1.39 +/- 0.59 (P = 0.021, Mann-Whitney). CONCLUSIONS p53 immunoreactivity and high proliferative activity in the epithelium overlying the pterygium are not associated with recurrence of pterygium.

Limbal cell autograft transplantation for severe ocular surface disorders

Abstract · Background: Limbal cell transplantation may improve the visual outcome after chemical trauma and ocular surface diseases. · Methods: Nine eyes of nine consecutive patients (eight males and one female, age 9–60 years), underwent limbal autograft transplantation (LAUT). In five cases LAUT was done for severe chemical burns in the acute stage (group 1). In four patients with old chemical trauma LAUT was performed years after the trauma (group 2). Penetrating keratoplasty (PKP) was carried out within 6 months after LAUT in three patients of group 2. Preoperatively, the visual acuity in all the patients except one was counting fingers. Postoperatively, patients were treated with topical antibiotics, topical corticosteroids and oral steroids. Oral cyclosporin was used after penetrating keratoplasty. · Results: No complications were observed during the surgical procedure. Postoperatively, the epithelialization was complete between days 7 and 12. The inflammatory response subsided within 3 months and the stromal neovascularization decreased. Visual acuity improved in all the nine cases, ranging from 6/6 to 6/30. The decreased visual acuity was due to corneal haze, scars and vascularization. Following PKP, the three grafts remained clear with intact epithelium. No complications were observed during the follow-up period from 7 to 60 months. · Conclusions: Limbal cell transplantation is an efficacious procedure for rehabilitation of visual acuity after severe chemical trauma.

Charged nanoparticles delivery to the eye using hydrogel iontophoresis.

Ocular iontophoresis has been investigated for many years as a non-invasive technique for enhancing ionized drug penetration through ocular tissues. In this study we assessed the penetration of charged fluorescent nanoparticles into rabbit eyes using hydrogel iontophoresis. Particle distribution into ocular tissues and penetration efficiency of negative nanoparticles compared with positive nanoparticles was also evaluated. Cathodal and anodal iontophoretic administrations were performed using polyacrylic hydrogels loaded with charged nanoparticle suspension (20-45 nm), applying a current intensity of 1.5 mA for 5 min onto the cornea and sclera. At pre-set time points post treatment, eyes were dissected and tissues were evaluated for fluorescence intensity. Strong fluorescence evidence was observed at anterior and posterior ocular tissues. Negative particle distribution profile revealed fast uptake into the outer ocular tissues, within 30 min post treatment, followed by particle migration into the inner tissues up to 12 h post treatment. The positively charged particles demonstrated better penetration abilities into inner ocular tissues compared to the negatively charge particles. This work provides an opening for the development of a new ocular therapeutic pathway using iontophoresis of extended release drug-loaded charged nanoparticles.

Iontophoresis–gentamicin delivery into the rabbit cornea, using a hydrogel delivery probe

PURPOSE To evaluate the efficacy of penetration of gentamicin into the cornea of rabbits using iontophoresis with a hydrogel-gentamicin containing probe. METHODS Eight of 10 groups (groups 3-10) of 6 rabbits (one eye per rabbit), underwent corneal iontophoresis using soft stable hydroxyethyl methacrylate hydrogel discs (80% water content) loaded with gentamicin sulphate which were mounted on an iontophoresis probe. The studied current intensities were 0, 0.1, 0.3 and 0.6 mAmp, and the durations of iontophoresis were 10 and 60 sec. Two control groups received 1.4% topical drops of gentamicin every 5 min for 1 hr (group 1) or sub-conjunctival injection of 10 mg gentamicin (group 2). Following sacrifice, aqueous humour was taken, corneas were excised, and gentamicin concentration was determined in aqueous humour and cornea samples. RESULTS Post-iontophoresis, the concentration of gentamicin in the corneas ranged from high (88.60 +/- 38.64 microg ml(-1)) to very low (0.10 +/- 0.89 microg ml(-1)). Both the control groups and those rabbits treated with current intensity of 0.1 mAmp or greater obtained therapeutic gentamicin levels in the corneas. Use of iontophoresis for 60 sec or current intensity greater than 0.1 mAmp obtained corneal gentamicin levels not different from sub-conjunctival injection. Application of current intensity of 0.1 mAmp or greater gave corneal gentamicin concentrations comparable to topical application of the drug, except when 0.6 mAmp were used for 60 sec (p = 0.05). Increasing current intensity or duration of iontophoresis significantly increased (p = 0.001 for both) gentamicin penetration into the cornea. Current intensity had more influence (Beta2 = 0.40) than duration (Beta2 = 0.13) on drug penetration. A significant interaction was found between the duration of iontophoresis and the current intensity. Very small or no concentrations of the drug were discovered in the anterior chambers of rabbits. CONCLUSIONS Iontophoresis using hydrogel-gentamicin probe may deliver therapeutic concentrations of gentamicin into the cornea.

Ex-vivo permeation study of indomethacin from a submicron emulsion through albino rabbit cornea

Abstract Indomethacin was incorporated in an original emulsion formulation stabilized by a combination of phospholipids and an amphoteric surfactant, lauroamphodiacetate. The solubility of indomethacin in the various emulsion phases was pH-dependent. The pH of the emulsion was adjusted to 3.8 in order to promote localization of the drug in the oil phase and prevent drug ionization. Ionization would increase drug aqueous solubility and result in indomethacin precipitation. Optimal manufacturing conditions were identified yielding an emulsion with a mean droplet size of 110±20 nm and a zeta potential value of −50 mV. The emulsion was found to be chemically and physically stable for more than 5 months at 4°C. The results of the ocular tolerance study in rabbit eye indicated that hourly administration of the emulsion vehicle was well tolerated without any toxic or inflammatory response to the ocular surface during the 5 days of the study. Scanning electron microscopy revealed a normal corneal surface resembling that of the animals treated with physiological saline. The penetration rate of indomethacin through excised rabbit eye cornea from the emulsion and from a marketed product (Indocollyre®) were determined and compared using a novel mounted corneal diffusion assembly. It was shown that the apparent corneal permeability coefficient of indomethacin incorporated in the emulsion was 3.8 times greater than that of indomethacin in the marketed aqueous solution. The increase in corneal drug permeation could be attributed to various causes that are discussed in the manuscript.

Evaluation of a positively charged submicron emulsion of piroxicam on the rabbit corneum healing process following alkali burn.

The effect of piroxicam in three different formulations was tested on rabbits for 28 days in an alkali burn model. The ulceration healing process was determined by evaluating the severity of the burn (scored from 0 to 5), and the re-epithelization healing process was measured by the area of the defects. The results indicated that the piroxicam positively charged submicron emulsion was the most effective formulation in lowering the ulcerative cornea score while the piroxicam positively charged emulsion and the blank emulsion were more effective in promoting the re-epithelization healing process. The piroxicam solution elicited the slowest healing re-epithelization rate after 28 days and was unable to complete the entire healing process. The new positively charged submicron emulsion formulation of piroxicam had a pronounced effect on both the ulceration rate and epithelial defects in the management of corneal alkali-burning.