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Biomedical subjects

Stefano Bonini

Publications and source records attributed to Stefano Bonini.

At least 19 recordsLinked to original sources
PubMed2006

Conjunctival expression of thymosin-beta4 in vernal keratoconjunctivitis.

PURPOSE: Thymosin-beta4 (Tbeta4) is a small actin-sequestrating peptide that modulates inflammation and healing in different tissues. The aim of this study was to investigate the molecular and biochemical expression of Tbeta4 in the healthy conjunctiva and in the conjunctiva of patients with vernal keratoconjunctivitis (VKC), a severe allergic eye disease characterized by chronic inflammation and marked tissue remodeling. METHODS: Conjunctival tissues, obtained from seven VKC patients and five sex/age-matched healthy subjects, were evaluated for Tbeta4 expression by relative real time-PCR and light/confocal microscopy. The distribution patterns of Tbeta4 in conjunctival sections as well as Tbeta4 expression by mast cells (AA1), eosinophils (EG2), and fibroblast/myofibroblasts (Thy1/alpha-SMA) in VKC tissues was also evaluated by double immunofluorescence. RESULTS: Compared to healthy specimens, Tbeta4 mRNA appeared upregulated in VKC conjunctival biopsies as detected by real-time PCR. In the healthy conjunctiva, Tbeta4 protein expression was confined to the conjunctival epithelium, while a weak staining was observed in the stroma. In VKC conjunctival sections, Tbeta4 immunoreactivity was selectively increased in the stroma where, by confocal analysis of VKC papillary formations, Tbeta4 appeared to be mostly localized in eosinophils and activated fibroblasts/myofibroblasts. CONCLUSIONS: Our data provides the first evidence of Tbeta4 expression in the conjunctival tissues. The upregulation of both Tbeta4 mRNA and protein in the conjunctival stroma, and its peculiar localization in eosinophils and myofibroblasts populating VKC lesions, suggest a possible role for Tbeta4 in tissue inflammation and remodeling occurring in VKC.

Adolescent↗
PubMed2006

Conjunctival mucin deficiency in complete androgen insensitivity syndrome (CAIS).

Sex steroid hormones are essential for a healthy ocular surface and the androgen receptor impairment found in patients with complete androgen insensitivity syndrome (CAIS) has been described to cause meibomian gland dysfunction and functional dry eye for lipid tear film layer instability. However, it has not been reported if the mucous layer is also affected. A 37-year-old CAIS patient with persistent symptoms of dry eye underwent ophthalmological examination and was evaluated for qualitative and quantitative tear function tests and conjunctival cytology. Samples obtained from the conjunctival epithelium were stained for histology and immunohistochemistry and compared with three age-matched female controls. Western blot and relative real-time RT-PCR for MUC1 and MUC5AC were also performed on these samples. Immunohistochemistry, Western blot and relative real-time RT-PCR showed a decrease in the expression of MUC1 and MUC5AC in CAIS. Changes in the tear film mucous layer were accompanied by a reduction in the tear film break up time test. This is the first report describing mucous layer alteration associated with androgen receptor impairment. Decreased mucin levels contribute in explaining the tear film instability in CAIS and should be considered an additional cause of dry eye in sex steroid hormone pathology.

Adult↗
PubMed2006

Nerve growth factor eye drop administrated on the ocular surface of rodents affects the nucleus basalis and septum: biochemical and structural evidence.

It has been shown that conjunctivally applied NGF in rats can reach the retina and optic nerve. Whether topical eye NGF application reaches the central nervous system is not known. In the present study, we have addressed this question. It was found that topical eye NGF application affects brain cells. Time-course studies revealed that repeated NGF application leads to high concentration of this neurotrophins after 6 h and normal levels after 24 h. Our results also showed that topical eye application of NGF causes an enhanced expression of NGF receptors and ChAT immunoreactivity in forebrain cholinergic neurons, suggesting that ocular NGF application could have a functional role on damaged brain cells. The present findings suggest that eye NGF application can represent an alternative route to prevent degeneration of NGF-receptive neurons involved in disorders such as Alzheimer and Parkinson.

Afferent Pathways↗
PubMed2006

Hyperosmolar conjunctival provocation for the evaluation of nonspecific hyperreactivity in healthy patients and patients with allergy.

BACKGROUND: Tissue hyperreactivity of target organs to nonspecific stimuli is known to be an important factor in influencing the clinical picture of allergic disease. OBJECTIVE: To identify the sensitivity and specificity of a hyperosmolar conjunctival provocation test in predicting conjunctival hyperreactivity and to relate this reactivity to the presence of ocular discomfort in subjects with and without allergy. METHODS: In 50 healthy patients and 19 patients with allergic conjunctivitis during remission phase, symptoms of ocular discomfort triggered by nonspecific stimuli were identified and graded with a discomfort score. Subjects were then challenged with a glucose solution at increasing concentrations (from 10% to 50%). The glucose concentration that elicited 2+ conjunctival hyperemia was considered the provoking dose. The response to this hyperosmolar provocation in subjects with ocular discomfort was compared with that of asymptomatic subjects. Sensitivity and specificity of the test in predicting conjunctival hyperreactivity were analyzed. RESULTS: Six of 50 healthy subjects and 12 of 19 subjects with allergy complained of ocular discomfort after exposure to nonspecific stimuli. The hyperosmolar provocation test discriminated between subjects with and without ocular discomfort (mean provoking dose: 39.5% +/- 5% and 47.5% +/- 5% glucose, respectively; P < .001). Forty percent glucose was the optimal threshold dose that demonstrated the highest sensitivity and specificity for prediction of conjunctival hyperreactivity. Discomfort scores were significantly related to provoking dose values (P < .05). CONCLUSION: This study provides a standardized procedure to detect nonspecific conjunctival hyperreactivity independent of underlying atopy. CLINICAL IMPLICATIONS: Hyperosmolar provocation test may be useful for identifying conjunctival hyperreactivity in subjects with and without allergy with a history of ocular discomfort.

Adolescent↗
PubMed2006

Nerve growth factor effect on human primary fibroblastic-keratocytes: possible mechanism during corneal healing.

In response to corneal injury, cytokines and growth factors play a crucial role by influencing epithelial-stromal interaction during the healing and reparative processes which may resolve in tissue remodeling and fibrosis. While transforming growth factor-beta1 (TGF-beta1) is considered the main profibrogenic modulator of these process, recently the nerve growth factor (NGF) appears as a pleiotropic modulator of wound-healing and inflammatory responses. Interestingly in the cornea, where NGF, trkA(NGFR) and p75(NTR) are expressed by epithelial cells and keratocytes, the NGF eye-drop induces the healing of neurotrophic or autoimmune corneal ulcers. During corneal healing, quiescent keratocytes are replaced by active fibroblast-like keratocytes/myofibroblasts. While the NGF effect on epithelial cells has been investigated, no data are reported for NGF effects on fibroblastic-keratocytes, during corneal healing. NGF, trkA(NGFR) and p75(NTR) were found expressed by fibroblastic-keratocytes. NGF was able to induce fibroblastic-keratocyte differentiation into myofibroblasts, migration, Metalloproteinase-9 expression/activity and contraction of a 3D collagen gel, without affecting their proliferation and collagen production. These data also show a two-directional control of fibroblastic-keratocytes by NGF and TGF-beta1. To sum up, the findings of this study indicate that NGF can modulate some functional activities of fibroblastic-keratocytes, thus substantiating the healing effects of NGF on corneal wound-healing.

Cell Differentiation↗
PubMed2006

Chemokine receptor CCR5 expression in conjunctival epithelium of patients with dry eye syndrome.

OBJECTIVE: To characterize chemokine receptor CCR5 expression on the conjunctival epithelium in dry eye syndromes. METHODS: Conjunctival impression cytology samples were obtained from normal subjects (n = 15) and patients with dry eye syndrome (n = 45). Cells were harvested from impression cytology samples, and flow cytometry was performed to quantitatively analyze the cell surface expression of chemokine receptor CCR5. Characterization of CCR5-positive cells was done by 2-color flow cytometry using fluorescein-conjugated anti-CCR5 and phycoerythrin-conjugated anti-CD45 antibodies (where CD45 is a marker for bone marrow-derived cells). To study CCR5 messenger RNA transcripts, real-time polymerase chain reaction was done on RNA isolated from the impression cytology samples of normal subjects (n = 5) and patients with dry eye syndrome (n = 14). RESULTS: We found significant up-regulation in cell surface expression of CCR5 in patients with both aqueous tear-deficient and evaporative forms of dry eye syndrome (P<.001). The real-time polymerase chain reaction results (for messenger RNA) corroborated the flow cytometry data (for protein). The majority of the cells expressing CCR5 were non-bone marrow-derived resident epithelial cells of the conjunctiva. CONCLUSION: Our findings suggest that CCR5 up-regulation is significantly associated with dry eye syndrome-associated ocular surface disease. Clinical Relevance Chemokine receptor CCR5 or its ligands may serve as useful targets for modulation of tissue immunoinflammatory responses in dry eye syndromes.

Adult↗
PubMed2005

Molecular basis for keratoconus: lack of TrkA expression and its transcriptional repression by Sp3.

Keratoconus is the most common corneal dystrophy that leads to severe visual impairment. Although the major etiological factors are genetic, the pathogenetic mechanism(s) is unknown. No medical treatments exist, and the only therapeutic approach is corneal transplantation. Recent data demonstrate the involvement of nerve growth factor (NGF) in trophism and corneal wound healing. In this study, we investigated alterations in the NGF pathway in keratoconus-affected corneas and found a total absence of the NGF-receptor TrkA (TrkA(NGFR)) expression and a decreased expression of NGF and p75(NTR). The absence of TrkA(NGFR) expression was associated with a strong increase in the Sp3 repressor short isoform(s) and a lack of the Sp3 activator long isoform. Sp3 is a bifunctional transcription factor that has been reported to stimulate or repress the transcription of numerous genes. Indeed, we found that Sp3 short isoform(s) overexpression in cell culture results in a down-regulation of TrkA(NGFR) expression. We suggest that an imbalance in Sp transcription-factor isoforms may play a role in controlling the NGF signaling, thus contributing to the pathogenesis of keratoconus. This mechanism for the transcriptional repression of the TrkA(NGFR) gene can provide the platform for the development of a therapeutic strategy.

Base Sequence↗
PubMed2005

Nerve growth factor modulates in vitro the expression and release of TGF-beta1 by amniotic membrane.

PURPOSE: To determinate the basal production of NGF and TGF-beta1 by amniotic membrane (AM) and to verify the presence of NGF receptors (trkA(NGFR) and p75(NTR)) in AM. Thereafter, to evaluate in an in vitro model if increasing concentrations of NGF are able to stimulate AM to produce and release TGF-beta1. METHODS: Immunohistochemistry, ELISA, in situ hybridization and PCR analysis for NGF, TGF-beta1, trkA(NGFR) and p75(NTR) were performed to study their presence in AM. Amniotic membranes were stimulated in vitro with increasing concentrations of NGF. After 24 h, the amount of TGF- beta1 in the AM tissue and in the culture medium was investigate. RESULTS: AM expressed TGF-beta1, NGF, trkA(NGFR) and p75(NTR) mRNAs and proteins and released basal amounts of NGF and TGF-beta1 in the medium. Stimulation of AM by addition of NGF induced a significant (P<0.05) increase of TGF-beta1 mRNA/protein in AM tissue as well as a release of TGF-beta1 protein into the culture medium, in a dose-dependent fashion. CONCLUSIONS: This study demonstrates that amniotic membrane releases basal amounts of NGF and TGF-beta1 and expresses NGF receptors . Moreover, the addition of NGF to the culture medium stimulates the amniotic membrane to significantly increase the production and release of TGF-beta1.

Amnion↗
PubMed2005

Clinical and cytological findings in limbal stem cell deficiency.

PURPOSE: To evaluate and relate the clinical (including corneal sensitivity and tear function) and cytological (presence of goblet cells and cytokeratin 3- and 19-positive cells) features of limbal stem cell deficiency (LSCD). METHODS: Twenty-nine patients (44 eyes) with a clinical diagnosis of LSCD participated in this study. Corneal signs (epithelial alterations, superficial neovascularisation and stromal scarring) and cytological findings (presence of goblet cells and cytokeratins 3 and 19) were evaluated and scored (from 0 to 3) from each of the five corneal sectors. Corneal sensitivity (Cochet-Bonnet aesthesiometer) and tear function (Schirmer test and BUT) were also assessed. Cytological scores were correlated statistically with both corneal signs and sensitivity values. RESULTS: Cytokeratin 19-positive cells were found in 82% of corneal impression cytology samples, while goblet cells were identified in only 59% of these same samples. Cytokeratin 3-positive cells were present in 61% of LSCD eyes and in all unaffected eyes. Corneal sensitivity was significantly decreased in affected eyes compared with contralateral, healthy eyes (1.6+/-1.7 cm versus 5.7+/-0.3 cm). Tear function tests did not show significant changes. In LSCD eyes, goblet cells and cytokeratin 19-positive cells on the corneal surface were significantly correlated to corneal epithelial alterations and to corneal superficial neovascularisation (p<0.001). Corneal cytokeratin 3-positive cells were inversely related to epithelial alterations (p=0.003). Corneal sensitivity was decreased in corneal sectors with epithelial alterations (p<0.001), neovascularisation (p<0.001) and stromal abnormalities (p=0.049), and was indirectly related to the presence of goblet cells (p=0.005) and cytokeratin 19-positive cells (p<0.001). CONCLUSION: This study confirmed the importance of cytological tests in the diagnosis of LSCD. Furthermore, the absence of goblet cells may not exclude corneal conjunctivalisation as demonstrated by cytokeratin 19 immunostaining. Lastly, corneal conjunctivalisation was associated with zone-specific impairment in corneal sensitivity.

Adolescent↗
PubMed2005

Efficacy of topical nerve growth factor treatment in dogs affected by dry eye.

BACKGROUND: Preliminary data show that nerve growth factor (NGF) may improve tear production in humans. We evaluated the efficacy of topical NGF treatment in dogs who developed dry eye after the excision of the third eyelid lacrimal gland. METHODS: English Bulldogs (2- to 6-year-old males and females) that had undergone the surgical removal of the prolapsed lacrimal gland of the third eyelid in both eyes at the age of 3-6 months developed chronic keratoconjunctivitis sicca associated with a decrease of Schirmer tear test I values after at least 1 year. One eye, randomly selected, of each dog was treated twice daily with 100 microl of NGF ointment for 1 month, while the fellow eye was used as control and treated with the ointment vehicle only. At baseline and after 1 month of NGF treatment the following examinations were performed: corneal evaluation by slit lamp, fluorescein staining, Schirmer tear test I, tear ferning test, corneal esthesiometry by cotton swab and conjunctival impression cytology. RESULTS: Topical application of NGF caused a significant improvement of all the evaluated parameters compared with baseline values. In contrast, in the control eyes there was no significant difference between the values measured before and after treatment. In particular, after NGF treatment superficial punctate keratopathy was resolved, corneal haze was reduced from stage 4 to stage 2 and Schirmer test values increased (17.2+/-1.7 mm/min vs 4.5+/-1.3 mm/min; p<0.05), as did the tear mucous component (as demonstrated by ferning test: 2.0+/-0.0 vs 4.0+/-0.0; p<0.05); conjunctival impression cytology evaluation demonstrated the presence of numerous mucous filaments and a significant increase in conjunctival goblet cell density (102.7+/-68.3 vs. 18.2+/-14.3 cell x field; p<0.05). Topical NGF treatment improved corneal sensitivity in two of three eyes. CONCLUSIONS: This open study suggests that topical application of NGF may enhance the production and functional characteristics in tear film, with an improvement of ocular surface signs in dogs with surgically induced dry eye. These results suggest the possibility of performing further, larger, controlled studies.

Administration, Topical↗
PubMed2005

Expression of Toll-like receptors in healthy and allergic conjunctiva.

PURPOSE: To investigate the expression of toll-like receptors (TLRs) in healthy and active vernal keratoconjunctivitis (VKC) conjunctiva. DESIGN: Comparative nonrandomized interventional study. PARTICIPANTS: Nine patients with vernal keratoconjunctivitis and 10 healthy subjects. METHODS: Normal and VKC conjunctiva were evaluated for TLR-2, TLR-4, and TLR-9 mRNA and proteins by relative real-time polymerase chain reaction and immunohistochemistry. Colocalization of TLRs was further evaluated by confocal microscopy and double-labeled immunofluorescence in T cells (CD4), mast cells (AA1), and eosinophils (EG2). MAIN OUTCOME MEASURES: Identification of TLRs at molecular and biochemical levels and identification of inflammatory cells that coexpress these receptors. RESULTS: Toll-like receptor 2, TLR-4, and TLR-9 were expressed in normal and VKC conjunctival epithelium and stroma, both with a more intense immunoreactivity in the stroma. In VKC tissues, TLR-4 was upregulated, TLR-9 was downregulated, and TLR-2 was slightly decreased relative to normal tissues. Toll-like receptor 4 was localized on CD4+ lymphocytes, eosinophils, and mast cells, whereas lymphocytes and eosinophils, but not mast cells, expressed TLR-9. CONCLUSIONS: Toll-like receptors are expressed in healthy and allergic conjunctiva. The upregulation or downregulation of these receptors and their presence on inflammatory cells suggest a role of TLRs in VKC.

Adolescent↗
PubMed2005

Effect of exogenous administration of nerve growth factor in the retina of rats with inherited retinitis pigmentosa.

NGF is implicated in retinal damage regression. To study whether this is a direct effect or an effect mediated by NGF on other endogenous biological mediators, we investigated the effect of exogenous administration of NGF in RCS rats affected by retinitis pigmentosa. We found that NGF administration exerts a rescue effect on photoreceptors in this animal model. NGF injection enhances brain-derived neurotrophic factor, beta-fibroblast growth factor, transforming growth factor-beta, vascular endothelial factor and neuropeptide-Y. This suggests that NGF has an effect on RCS rat retina, probably also through the stimulation of other biological mediators produced and released in the retina.

Animals↗
PubMed2005

Pharmacokinetics of conjunctivally applied nerve growth factor in the retina and optic nerve of adult rats.

PURPOSE: Nerve growth factor (NGF) has been shown to inhibit retinal ganglion cell (RGC) degeneration when injected intraocularly in animal models of ocular hypertension, optic nerve transaction, and ischemia. The present study sought to establish the bioavailability of topical NGF to the retina and optic nerve in rats. METHODS: Autoradiography was performed to evaluate whether exogenous (125)I-labeled NGF reaches the retina and optic nerve when applied topically to the rat conjunctiva. To quantify NGF levels, a highly specific immunoenzymatic test (ELISA) was performed on the retina, optic nerve, lens, sclera and serum of rats at different time points after administration of NGF (1-500 microg/mL). The physiological activity of topically applied NGF was evaluated by determining retinal brain-derived neurotrophic factor (BDNF) protein and mRNA levels by ELISA and RT-PCR, respectively. RESULTS: After topical conjunctival administration of NGF, high levels were detected in ocular tissues, including the retina and optic nerve, showing a peak increase 6 hours after administration at a concentration of 200 mug/mL. NGF treatment was associated with an increase in BDNF protein and mRNA levels in rat retina. CONCLUSIONS: These data demonstrate the bioavailability of NGF to the retina and optic nerve in rats when administered topically. These findings justify investigating the clinical effects of topical NGF therapy for treatment of posterior segment diseases.

Administration, Topical↗
PubMed2004

Demographic and clinical factors associated with development of brimonidine tartrate 0.2%-induced ocular allergy.

PURPOSE: To identify demographic and clinical characteristics associated with the development of brimonidine tartrate 0.2%-induced ocular allergy. PATIENTS AND METHODS: In this retrospective study, 133 patients affected by primary open-angle, pigmentary, narrow angle, or pseudo-exfoliative glaucoma and treated with brimonidine tartrate 0.2% were divided into two groups: allergic and non allergic to brimonidine tartrate 0.2%. The distribution of demographic (age and sex), local (history of allergic conjunctivitis, previous eye-drop ocular allergy, use of other concurrent topical medications, amount of topical medications previously used, use of contact lenses, and tear film production), and systemic (history of systemic allergies and use of systemic drugs) factors was evaluated by comparing the brimonidine tartrate 0.2% allergic and the non-allergic groups. RESULTS: In this study, 13.5% of patients (18 of 133) developed brimonidine ocular allergy generally within two weeks from the beginning of treatment (mean time 14.8 +/- 17.9 days). The brimonidine tartrate 0.2% allergic group showed a significantly higher frequency of history of ocular allergy to eye-drops (P = 0.048) and to topical beta-blockers (P = 0.019) when compared with the brimonidine tartrate 0.2% non-allergic group. Moreover, the allergic group showed a decreased tear film production (P = 0.044). CONCLUSION: This study showed that history of eye-drop allergies and reduction of tear film production were more frequently associated with the development of brimonidine tartrate 0.2%-induced ocular allergy.

Adrenergic alpha-Agonists↗
PubMed2004

Nerve growth factor and the immune system: old and new concepts in the cross-talk between immune and resident cells during pathophysiological conditions.

PURPOSE OF REVIEW: This review provides an overview of nerve growth factor and its involvement in the immune system. Furthermore, recent data are provided revealing new important insights into the mechanisms of action of nerve growth factor in allergic reaction. RECENT FINDINGS: Recent studies on the effects of nerve growth factor on the immune cells involved allergic reaction, and on the potential role of nerve growth factor in tissue remodelling are presented. SUMMARY: Nerve growth factor has an extended function from the nervous system to a wide range of activities in the immune system. Several papers have highlighted the role of the factor in allergic inflammation. This review describes old and new concepts of nerve growth factor in the immune system: the relation between nerve growth factor and the main cells taking part in allergic inflammatory disorders, structural cells, mediators and cytokines/chemokines, as well as the mechanisms leading to nerve growth factor increase. Understanding these complex mechanisms will introduce new therapeutic approaches for nerve growth factor in the immune system, in addition to those already established in the nervous system.

Cell Communication↗