Barbara Stampachiacchiere

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)

Differential modulatory effect of NGF on MHC class I and class II expression in spinal cord cells of EAE rats.

Nerve growth factor (NGF) undergoes significant changes in the central nervous system (CNS) of patients affected by multiple sclerosis (MS) and of rats with experimental allergic encephalomyelitis (EAE). The major histocompatibility complex (MCH) class I and class II antigens are molecules that play a pivotal role in these neuro-inflammatory disorders. The aim of this study was to investigate the role of NGF on MCH class I and class II antigens in spinal cords cells of EAE rats. It was found that the administration of NGF in EAE rats enhances MHC-I, IFN-gamma receptor and interferon regulatory factor-1 expression on the neurons but not in the glial cells, while NGF decreased MHC class II antigen in the glial cells. NGF administration into the brain of EAE rats has no effect on TNF-alpha expression. The present findings suggest that NGF may have a regulatory function in spinal cord cells during tissue inflammation.

Human idiopathic epiretinal membranes express NGF and NGF receptors.

Purpose: Glial cells and fibroblasts (FBs) play a key role in epiretinal membrane (ERM) development and progression. Myofibroblasts (myoFBs), arising from these cells, can lead to the hypertrophic scars and tissue contraction observed in ERMs. Nerve growth factor (NGF) and transforming growth factor-&bgr;1 (TGF-&bgr;1) play a crucial role in FB activities. Therefore, the authors evaluated myoFBs in ERMs and NGF, trkANGFR and p75NTR expression, as well as TGF-&bgr;1/TGF-&bgr;RII levels in both ERMs and vitreous. Methods: Eight idiopathic ERMs and vitreous were obtained from patients at the time of vitrectomy for macular pucker. Ten control vitreous were from donors. Biochemical and molecular analyses were performed to identify &agr;-smooth muscle actin (&agr;-SMA, a defined myoFB marker), NGF, trkANGFR/p75NTR, and TGF-&bgr;1/TGF-&bgr;RII. Results: Every idiopathic ERM displayed &agr;-SMA positive myoFBs, expressing NGF, trkANGFR, and p75NTR. ERM vitreous showed a significant decrease in NGF protein coupled with a TGF-&bgr;1 increase. In addition, vitreous cells showed an increase in trkANGFR/p75NTR mRNA associated with a decrease in TGF-&bgr;RII mRNA. Conclusions: Idiopathic ERMs were characterized by myoFBs. The expression of NGF, trkANGFR, and p75NTR in local myoFBs associated with changes in ERM vitreous NGF suggests an involvement of NGF, as previously reported for TGF-&bgr;1, in the evolution and myoFB-mediated contractile activity of ERMs.

Toll-like receptors and the eye.

Purpose of reviewThis review will describe the structure, expression/distribution and functional activity of Toll-like receptors, in particular in the ocular structures. It will also discuss innate and adaptive immune responses, by exploring the possible modulation/regulation of innate and adaptive immunity by Toll-like receptors, in view of recent findings observed in the ocular surface. Recent findingsCurrent knowledge indicates that Toll-like receptors represent essential elements in host defence against pathogens, a prerequisite to the induction of adaptive immune responses. The expression/distribution of Toll-like receptors in the healthy eye highlights the possible function of Toll-like receptors in both innate and adaptive responses during pathological conditions of the ocular surface. SummaryRecent findings have greatly increased the knowledge of the possible role of Toll-like receptors in innate and adaptive immune responses. Toll-like receptors seem to play different roles in a wide range of activities of the immune system, and might represent an exclusive link between innate and adaptive responses under pathological conditions. Recent studies in ophthalmology have highlighted the role of Toll-like receptors in infections (keratitis) as well as in allergic states of the ocular surface. This review thus describes the relationship between Toll-like receptors and the main immune/structural cells taking part in inflammatory disorders. Understanding the complex mechanisms underlying Toll-like receptor localization and function will provide additional data that might help devise novel therapeutic approaches involving Toll-like receptors and their agonists, in an attempt to modulate the biased immune system.

Effect of High-Dose Atorvastatin Reload on the Release of Endothelial Progenitor Cells in Patients on Long-Term Statin Treatment Who Underwent Percutaneous Coronary Intervention (from the ARMYDA-EPC Study)

Endothelial progenitor cells (EPCs) may concur to endogenous vascular repair. Previous studies have reported that statin treatment increases EPC levels. We investigated whether this occurs in patients on long-term statin treatment who underwent percutaneous coronary interventions (PCIs). In a phase A study, 53 patients (atorvastatin reload [AR] 80 mg 12 hours before + 40 mg 2 hours before PCI, n = 27; placebo [P], n = 26) were evaluated for EPC mobilization as CD45dim/CD34+/CD133+/KDR+ cell number by flow cytometry. Assays were run at randomization (12 hours before PCI, R), immediately before PCI (T0) at 8 (T8) and 24 hours (T24). In phase B study, 50 patients (AR, n = 25; P, n = 25) were evaluated for early colony formation by Hill colony forming unit (CFU) assay, with sampling at randomization and 24 hours later. In phase A, EPCs levels were similar at randomization between 2 arms (0.23% [0.14 to 0.54] of total events in AR vs 0.22% [0.04 to 0.37] in P group; p = 0.33). At PCI, EPC levels were higher in AR arm (0.42% [0.06 to 0.30] vs 0.19% [0.06 to 030]; p = 0.009). Higher EPC levels in AR group were also found at 8 and 24 hours. In phase B, EPC CFUs/well numbers at randomization were similar in the 2 arms (8 [6 to 12] in AR vs 12 [6 to 20] in P group, p = 0.109). EPC CFU/well at 24 hours became significantly higher in AR arm (17 [10 to 23] vs 5 [2 to 13], p = 0.002). In conclusion, high-dose AR before PCI in patients on long-term statin therapy promptly increases EPCs mobilization, which are capable of early colony formation and may contribute to cardioprotection.

Allergic bronchial airway inflammation in nerve growth factor (NGF)-deprived rats: Evidence suggesting a neuroimmunomodulatory role of NGF

In the present study, ovalbumin-sensitized/challenged rats were characterized by an nerve growth factor (NGF) increase in both serum and bronchial alveolar lavage fluid (BALF), but not in the lung. Exogenous administration of NGF or NGF-neutralizing antibodies did not modify immunoglobulin (IgE) and eosinophil parameters. In control rats, NGF administration did not induce increase of IgE or eosinophils in both BALF and lung. The present findings suggest that at least NGF does not act as a proper proinflammatory factor but most probably as a neuroimmune modulator molecule of the allergic state.

Molecular and biochemical expression of TLRs in human amniotic membrane: A comparative study of fresh and cryopreserved specimens

BackgroundTo assess the expression of toll-like receptors (TLRs) in human amniotic membrane (AM) specimens and compare this expression with those of AMs undergoing the standard preservation procedure (handling) for ocular surgery.MethodsHuman fresh (n = 10; five spontaneous and five cesarean) or handled (n = 5) AMs were analyzed for TLR gene and protein expression. Two pieces were obtained from each specimen, and subjected to molecular or biochemical analysis. Relative real-time PCR and SDS-PAGE were carried out according to standard procedures. The REST–ANOVA coupled analysis was used to compare the molecular and biochemical data.ResultsThe fresh membranes expressed all the TLRs (TLR1-10), with different gene expression as detected/evidenced by the Ct values, the intra-fresh group analysis showing that there was a variation of TLR expression whichvaried within the fresh membranes. The handled AMs retained the TLR expression after standard processing and preservation, but with a particular pattern which included a high TLR3/TLR4 and low TLR6 expression, when compared to the fresh membranes. The molecular data were confirmed by Western blot analysis.ConclusionsAM is routinely used in several ophthalmic surgical procedures, and notwithstanding its preservation procedure, AM is reported to favour wound healing and exert anti-angiogenic, anti-inflammatory, anti-scarring as well as anti-bacterial activities. The presence of TLRs in handled AM would imply that TLRs might be preserved in AMs used in ocular surgery. The findings herein described provide additional data concerning the presence of TLRs in cryopreserved AM, and suggest a possible contribution of AM in ocular surgery, via the innate immune response.

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.