Roberto Sgrulletta

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 reviewThis 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 findingsRecent 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. SummaryNerve 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.

In vivo characterization of doxycycline effects on tear metalloproteinases in patients with chronic blepharitis

Purpose Matrix metalloproteinases (MMPs) have a role in the pathogenesis of rosacea-associated chronic blepharitis. Doxycycline is largely used as a treatment for recalcitrant chronic blepharitis. It has been shown in vitro that doxycycline inhibits MMPs activation. The aim of this study was to investigate in vivo the effect of doxycycline in modulating MMPs in patients with chronic idiopathic blepharitis. Methods Eight patients (6 male, 2 female; mean age 45.7±17.5 years) were included in the study. Doxycycline (100 mg) was administered orally, twice a day, for 2 weeks and once a day for an additional 2 weeks. Clinical signs and symptoms were evaluated and scored (0–3) at baseline and after 4 weeks. Total sign (TSS) and total symptom (TSyS) scores were calculated. Tear samples and conjunctival impression cytologies were collected at baseline and after 4 weeks of treatment to evaluate MMP-9 and TIMP-1 expression and activity. Results An improvement in TSS (4.5±1.1 vs 2.7±1.5) and TSyS (6.6±1.3 vs. 3.1±1.9) was observed after 4 weeks, with significant amelioration of hyperemia, marginal blepharitis, and superficial punctuate keratopathy. Zymography revealed a decrease of MMP-9 activity after 4 weeks. MMP-9 mRNA and protein levels did not change, while an upregulation of TIMP-1 expression was observed. Conclusions This study suggests that 4-week treatment with doxycycline significantly improved symptoms and signs in patients with chronic blepharitis in association with a decrease in MMP-9 activity. Upregulation of TIMP-1 is proposed as a possible mechanism of MMP-9 inactivation. (Eur J Ophthalmol 2009; 19: 708–16)

Preservative-free diclofenac sodium 0.1% for vernal keratoconjunctivitis

PurposeThe aim of this study is to evaluate the efficacy of prolonged treatment with preservative-free diclofenac sodium 0.1% eye drops in patients with vernal keratoconjunctivitis (VKC).MethodsA prospective open study was performed in 22 patients with VKC treated with preservative-free diclofenac sodium 0.1% eye drops. Patients used the eye drops four times daily in both eyes for 120 days. Signs (papillae, hyperaemia and corneal lesions) and symptoms (itching, redness and photophobia) of the ocular surface were graded and statistically evaluated before and after treatment by a non-parametric test (Mann–Whitney U-test).ResultsForty per cent of the patients showed an improvement in their symptoms at the end of the treatment. Total signs and symptoms scores were significantly decreased at the end of treatment compared with the baseline values (from 6.13±1.45 to 0.81±0.90 and from 5.40±1.18 to 2.63±0.95, respectively; P<0.001). Significant decreases in conjunctival redness (P<0.001), itching (P<0.001) and photophobia (P<0.001 ) were observed at the end of treatment. Conjunctival hyperaemia was significantly reduced (P<0.001) at the end of treatment, while no significant differences were observed for corneal lesions and for papillary size. No patient showed exacerbation of the disease during the treatment.ConclusionVKC is a chronic disease that requires prolonged treatment to control the inflammatory process. Our preliminary study demonstrates the efficacy and safety of preservative-free diclofenac sodium 0.1% eye drops in controlling the signs and symptoms of VKC in prolonged treatment.

Upregulation of ICAM-1 expression in the conjunctiva of patients with chronic graft-versus-host disease.

Purpose To correlate conjunctival intercellular adhesion molecule 1 (ICAM-1) expression with cytologic and clinical findings of chronic graft-versus-host disease (GVHD). Methods Seven patients with chronic GVHD-related keratoconjunctivitis and five age-matched normal controls were recruited for the study. Clinical examination included medical history, visual acuity, evaluation of ocular signs and symptoms (scored from 0 to 3), corneal fluorescein staining (scored from 0 to 5 on the basis of the number of corneal sectors involved), Schirmer test type I, and break-up time (BUT). Impression cytology samples were collected from the nasal and inferior bulbar conjunctiva of patients and controls. Goblet cells were counted in three randomly selected fields and averaged. Immunofluorescent staining for ICAM-1 was carried out and the percentage of cells expressing the marker was evaluated. Results All patients showed signs and symptoms of keratoconjunctivitis sicca. Schirmer test type I and BUT were reduced (4.8±6.7 mm/5 min and 3.9±2.7 seconds, respectively). Goblet cells were significantly reduced in GVHD eyes with respect to normal eyes (65±30.5 and 192±16.9 cells/field respectively; p<0.001). Goblet cell number was directly related to Schirmer test values (p<0.01, rho=0.817) and inversely related to total sign score (p<0.01, rho=-0.939). ICAM-1 expression was increased in GVHD eyes with respect to normal controls, in which no staining was observed. ICAM-1 expression showed an inverse relation to goblet cell number (p<0.01, rho=-0.852) and Schirmer test values (p<0.01, rho=-0.926), and was directly correlated to total sign score (p<0.01, rho=0.982). Conclusions Conjunctival ICAM-1 expression is increased in GVHD patients. The severity of the disease is associated with tear parameters, goblet cell decrease, and inflammatory markers, such as ICAM-1.

Amniotic membrane transplantation associated with conjunctival peritomy in the management of Mooren's ulcer: A case report

Purpose To report the association of conjunctival peritomy with amniotic membrane transplantation (AMT) at the limbus with the exclusion of the central cornea in order to preserve visual function in one case of bilateral Moorens ulcer. Methods A 36-year-old man with bilateral Moorens ulcer was unresponsive to conventional therapy. Surgical procedure was performed on his right eye, at impending risk of corneal perforation. A 20 × 20 mm piece of amniotic membrane (AM) was prepared by performing a central hole of 7.5 mm diameter with a manual trephine. A 360° conjunctival peritomy was performed and the AM was placed with the epithelium side facing up and the central hole was sutured on the paracentral cornea. Results Two weeks after surgery, while the right eye showed improvement of signs and symptoms and unchanged best-corrected visual acuity (BCVA), the left eye showed a peripheral corneal perforation with prolapsed iris that required conjunctival flap. At 7 months of follow-up, the right eye showed no ocular inflammation, a reduction of the lipid-like peripheral corneal infiltrates, an increased stromal thickness, and an unchanged BCVA. The progression of corneal thinning in the left eye led the authors to perform AMT (as described) in the left eye as well. Five months after the AMT in the left eye, neither eye shows signs of disease progression, and neither requires further therapy. Conclusions Conjunctival peritomy associated with AMT may be an alternative surgical approach in the management of Moorens ulcers to control the inflammation and the progression of disease.

Allergic Conjunctivitis: Update on Its Pathophysiology and Perspectives for Future Treatment

The ocular surface mucosa is constantly exposed to allergens and, therefore, highly susceptible for developing allergic reactions. Allergic conjunctivitis is among the most common diseases faced by ophthalmologists and it has been traditionally divided in four categories based on clinical presentation: seasonal and perennial allergic conjunctivitis, the two milder forms, and vernal and atopic keratoconjunctivitis, the two most severe forms often characterized by corneal involvement. Although they may present with different grades of severity, all four classes of allergic conjunctivitis share common symptoms such as ocular redness, itching and tearing, and they are all characterized by inflammatory reaction and eosinophil infiltration of the conjunctiva. However, while seasonal and perennial allergic conjunctivitis are characterized by a well-defined pathogenesis, with a typical type I hypersensitivity reaction, vernal and atopic keratoconjunctivitis feature more complex pathogenetic mechanisms: in the former, specific sensitization is not found in many patients and the disease typically regresses with puberty; in the latter, patients have atopic dermatitis or eczema from childhood, but develop ocular symptoms only later in life. Considering the variety of clinical presentations and the diverse pathophysiology characterizing the different forms of allergic conjunctivitis, several therapeutic options are available and newer therapies with immunomodulatory agents are under consideration in recent and ongoing clinical trials. In this chapter we will describe in detail the pathophysiology of ocular allergy, the mechanisms of action of all the available treatments, and the perspectives for future therapies that have been introduced by recent findings in clinical and basic research studies.

Allergy and infections: Long-term improvement of vernal keratoconjunctivitis following viral conjunctivitis

Purpose Vernal keratoconjunctivitis (VKC) is a severe, chronic allergic inflammatory disease of the ocular surface poorly responsive to antiallergic treatments and possibly leading to permanent visual impairment. VKC, because of mast cell, eosinophil, and Th2-type inflammation, polyclonal IgE activation, and tissue remodeling, is considered to be a typical Th2-driven disease. Viral infection stimulates a Th1 type immune response, potentially attenuating allergen-induced inflammation. The purpose of this report is to describe the effect of viral keratoconjunctivitis in a patient with VKC. Methods The authors report on a patient with a severe form of VKC, poorly responsive to antiallergic treatments, who developed a viral keratoconjunctivitis. Signs, symptoms, and cytologic findings were recorded during the 5-year follow-up period. Results The authors observed a prompt and permanent improvement of signs and symptoms of the allergic condition after the viral infection. Conjunctival scraping confirms that the inhibition of the eosinophilic inflammation lasts at least for 5 years. Conclusions In this case, the viral infection seemed to induce a clinical recovery of allergic disease, suggesting that an immune deviation induced by Th1-polarizing agents may revert an ongoing Th2 inflammation.

Quiescent and Active Tear Protein Profiles to Predict Vernal Keratoconjunctivitis Reactivation

Objective. Vernal keratoconjunctivitis (VKC) is a chronic recurrent bilateral inflammation of the conjunctiva associated with atopy. Several inflammatory and tissue remodeling factors contribute to VKC disease. The aim is to provide a chip-based protein analysis in tears from patients suffering from quiescent or active VKC. Methods. This study cohort included 16 consecutive patients with VKC and 10 controls. Participants were subjected to clinical assessment of ocular surface and tear sampling. Total protein quantification, total protein sketch, and protein array (sixty protein candidates) were evaluated. Results. An overall increased Fluorescent Intensity expression was observed in VKC arrays. Particularly, IL1β, IL15, IL21, Eotaxin2, TACE, MIP1α, MIP3α, NCAM1, ICAM2, βNGF, NT4, BDNF, βFGF, SCF, MMP1, and MMP2 were increased in quiescent VKC. Of those candidates, only IL1β, IL15, IL21, βNGF, SCF, MMP2, Eotaxin2, TACE, MIP1α, MIP3α, NCAM1, and ICAM2 were increased in both active and quiescent VKC. Finally, NT4, βFGF, and MMP1 were highly increased in active VKC. Conclusion. A distinct “protein tear-print” characterizes VKC activity, confirming some previously reported factors and highlighting some new candidates common to quiescent and active states. Those candidates expressed in quiescent VKC might be considered as predictive indicators of VKC reactivation and/or exacerbation out-of-season.

NGF modulates trkANGFR/p75NTR in αsMA-expressing conjunctival fibroblasts from human ocular cicatricial pemphigoid (OCP)

Objective In a previous study, we reported the upregulation of Nerve Growth Factor (NGF) and trkANGFR expression in Ocular Cicatricial Pemphigoid (OCP), an inflammatory and remodeling eye disease. Herein, we hypothesize a potential NGF-driven mechanism on fibroblasts (FBs) during OCP remodeling events. To verify, human derived OCP-FBs were isolated and characterized either at baseline or after NGF exposure. Materials and Methods Conjunctival biopsies were obtained from 7 patients having OCP and 6 control subjects (cataract surgery). Both conjunctivas and primary FB cultures were characterised for αSMA, NGF and trkANGFR/p75NTR expression. Subcultures were exposed to NGF and evaluated for αSMA, NGF, trkANGFR/p75NTR expression as well as TGFβ1/IL4 release. For analysis, early and advanced subgroups were defined according to clinical parameters. Results OCP-conjunctivas showed αSMA-expressing FBs and high NGF levels. Advanced OCP-FBs showed higher αSMA expression associated with higher p75NTR and lower trkANGFR expression, as compared to early counterparts. αSMA expression was in keeping with disease severity and correlated to p75NTR. NGF exposure did not affect trkANGFR levels in early OCP-FBs while decreased both αSMA/p75NTR expression and TGFβ1/IL4 release. These effects were not observed in advanced OCP-FBs. Conclusions Taken together, these data are suggestive for a NGF/p75NTR task in the potential modulation of OCP fibrosis and encourages further studies to fully understand the underlying mechanism occurring in fibrosis. NGF/p75NTR might be viewed as a potential therapeutic target.

Age-Related Changes to Human Tear Composition

Purpose We characterize age-associated alterations in the expression of inflammatory mediators and tissue remodeling factors in human tears. Methods A total of 75 consecutive volunteers (32 male/44 female; 19-93 years) underwent clinical assessment of ocular surface status, ocular surface disease index (OSDI) grading and tear sampling. The volunteers were categorized into three groups: young (18-40 years), middle-aged (41-60 years), and old (>60 years). Total protein profiles and chip-based protein array evaluations were conducted to investigate the expression of 60 potential candidates, including pro-/anti-inflammatory mediators and tissue remodeling factors. Appropriate validations were performed using conventional assays. Multiple comparisons for regression between potential candidates and age were performed, as well as statistical analyses among the three age groups. Nonpooled samples were used for quantifications. Results Pearson analysis of chip-arrays identified 9 of 60 potential candidates. Specifically, IL-8, IL-6, and regulated on activation, normal T cell expressed and secreted (RANTES; P < 0.0083) protein as well as matrix metalloproteinase (MMP)-1, IL-3, and TNF-α (P < 0.05) correlated positively with aging. MIP-3β showed an opposite tendency. Western blot and ELISA analysis corroborated the array data. OSDI grading did not correlate with aging. Conclusions Dynamic changes to tear protein profiles occur with aging. Our study identifies the expression of IL-8, IL-6, RANTES, MMP-1, and MIP-3β as increasing with age. These select inflammatory and matrix remodeling factors may be relevant to the development of novel diagnostic tools and therapeutics in the context of age-related ocular surface disease.