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.

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 TGFß1/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 TGFß1/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.

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

BACKGROUND: To 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. METHODS: Human 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. RESULTS: The 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. CONCLUSIONS: AM 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.

Nerve growth factor modulates toll-like receptor (TLR) 4 and 9 expression in cultured primary VKC conjunctival epithelial cells.

PURPOSE: To investigate if nerve growth factor (NGF) might modulate toll-like receptor (TLR) 4 and 9 expression in primary cultures of vernal keratoconjunctivitis (VKC)-derived conjunctival epithelial cells (VKC-ECs). METHODS: Primary cultures of ECs were established from VKC (n=7) and healthy-control (n=5) conjunctival specimens. Primary untouched and short-term NGF-exposed VKC-ECs were analyzed for molecular (relative real-time PCR) and biochemical (confocal and fluorescence-activated cell sorting analysis of TLR4 and TLR9 expression. Data were compared to their untreated as well as stimulated healthy-control counterparts. Conditioned media were analyzed for interferon (IFN)-gamma, interleukin (IL)-4, IL-10, and IL-12 p40 secretion. RESULTS: Primary untouched VKC-ECs showed TLR4 increase and TLR9 decrease compared to their healthy-control counterparts. NGF exposure resulted in a strong upregulation of TLR4 and a moderate upregulation of TLR9 in few passages VKC-ECs. Both TLR4 and TLR9 upregulation occurred in a dose-dependent fashion and were supported by biochemical analysis. NGF triggered a dose-response release of IL-10 in VKC-ECs conditioned media, an effect not detected for IL-4, IL-12 p40, and IFN-gamma. CONCLUSIONS: Our data indicate that NGF is able to induce TLR4/TLR9 overexpression in VKC-ECs. These cells exhibited poor IL-4, IL-12 p40, and IFN-gamma responses to NGF, while a significant IL-10 decreased secretion was detected. The different NGF-induced TLR response between VKC and healthy-control conjunctival ECs as well as the different cytokine response might reflect a different pattern of cell activation according to the state of VKC.

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.

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-beta1 (TGF-beta1) play a crucial role in FB activities. Therefore, the authors evaluated myoFBs in ERMs and NGF, trkA(NGFR and p75(NTR) expression, as well as TGF-beta1/TGF-betaRII 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 alpha-smooth muscle actin (alpha-SMA, a defined myoFB marker), NGF, trkA(NGFR)/p75(NTR), and TGF-beta1/TGF-betaRII. RESULTS: Every idiopathic ERM displayed alpha-SMA positive myoFBs, expressing NGF, trkA(NGFR), and p75(NTR). ERM vitreous showed a significant decrease in NGF protein coupled with a TGF-beta1 increase. In addition, vitreous cells showed an increase in trkA(NGFR)/p75(NTR) mRNA associated with a decrease in TGF-betaRII mRNA. CONCLUSIONS: Idiopathic ERMs were characterized by myoFBs. The expression of NGF, trkA, and p75 in local myoFBs associated with changes in ERM vitreous NGF suggests an involvement of NGF, as previously reported for TGF-beta1, in the evolution and myoFB-mediated contractile activity of ERMs.

Preliminary evidence of the efficacy of probiotic eye-drop treatment in patients with vernal keratoconjunctivitis.

BACKGROUND: Probiotics have been shown to improve allergic inflammation. The aim of this study was to evaluate the efficacy of Lactobacillus Acidophilus eye-drops in controlling signs and symptoms of vernal keratoconjunctivitis (VKC). METHODS: Seven patients (mean age 11.8 +/- 4.3; five M, two F) with mild to moderate VKC were included in the study. Lactobacillus Acidophilus diluted in saline solution (2 x 10(8) CFU/ml) was administrated as eye-drops four times daily for 4 weeks in both eyes. Clinical signs (conjunctival hyperemia, chemosis, secretion, Trantas dots, superficial punctuate keratitis) and symptoms (itching, photophobia, burning, tearing) were evaluated and scored from 0 to 3 at baseline, after 2 and 4 weeks of treatment. Total sign (TSS) and symptom (TSyS) scores were calculated. Conjunctival impression cytology was performed in three patients at baseline and after 4 weeks of treatment, in order to evaluate the expression of ICAM-1 and TLR-4. RESULTS: In the six out of seven patients who completed the study, symptoms were significantly improved after both 2 weeks (TSyS: baseline 6.7 +/- 0.9 vs 4.1 +/- 1.2; p = 0.017) and 4 weeks (TSyS: baseline 6.7 +/- 0.9 vs 3.6 +/- 1.2, p = 0.011) of treatment. A significant improvement of clinical signs was observed after 4 weeks of treatment (TSS: baseline 7.5 +/- 1.6 vs 3.9 +/- 1.7, p = 0.034) but not after 2 weeks of treatment (TSS: baseline 7.5 +/- 1.6 vs 5.3 +/- 1.5; NS). In particular, photophobia was significantly reduced (2 +/- 0.6 vs 1 +/- 0.3; p = 0.023) at 2 weeks, while at 4 weeks the scores for itching (1.8 +/- 0.3 vs 1 +/- 0.3), tearing (1.6 +/- 0.4 vs 0.8 +/- 0.2), conjunctival hyperemia (2.3 +/- 0.2 vs 1.4 +/- 0.5) and chemosis (1.2 +/- 0.4 vs 0.4 +/- 0.4) were significantly lower compared to baseline. A down-regulation of ICAM-1 and TLR-4 was observed in two patients showing clinical improvement after 4 weeks of treatment. CONCLUSION: Our open pilot study showed that 1-month treatment with probiotic eye-drops improves signs and symptoms in patients with VKC. Additional double-blind controlled clinical trials with a larger sample of patients are needed to confirm the effects of topical Lactobacilli on VKC patients.

Nerve growth factor has a modulatory role on human primary fibroblast cultures derived from vernal keratoconjunctivitis-affected conjunctiva.

PURPOSE: To evaluate the role of nerve growth factor (NGF) in remodeling processes of vernal keratoconjunctivitis (VKC). VKC is a chronic inflammatory disorder of the conjunctiva and is characterized by marked tissue remodeling. NGF, a pleiotrophic factor with documented profibrogenic activities, is produced by inflammatory and structural cells populating the VKC conjunctiva and is increased in the serum and tears of VKC patients. METHODS: Primary cultures of VKC-derived fibroblasts (VKC-FBs) were exposed to increasing NGF concentrations (1-500 ng/ml) to evaluate and compare the expression of alpha-smooth muscle actin (alphaSMA, a defining myofibroblast marker), collagens (types I and IV), and metalloproteinases and tissue inhibitors (MMP9/TIMP1, MMP2/TIMP2) at the biochemical as well as molecular levels. RESULTS: Endogenous NGF was increased in the VKC-FB supernatant, as compared to healthy-FB supernatant. VKC-FBs expressed alphaSMA and increased types I and IV collagens. VKC-FBs, and in particular all alphaSMA positive cells, expressed both trkA(NGFR) and p75(NTR), while healthy-FBs only expressed trkA(NGFR). Exogenous NGF did not change alphaSMA expression, while alphaSMA expression was enhanced by specific neutralization of p75(NTR). NGF (10 ng/ml) exposure significantly decreased type I collagen expression, without affecting type IV collagen, and increased MMP9mRNA and protein. CONCLUSIONS: The autocrine modulation of differentiation and response of VKC-FBs to NGF exposure with downregulation of type I collagen and upregulation of MMP9 expression supports a relevant role for NGF in tissue remodeling of VKC.

Nerve growth factor and tissue repair remodeling: trkA(NGFR) and p75(NTR), two receptors one fate.

This review deals with the role of nerve growth factor (NGF) in healing process as a result of injury. The role of both trkA(NGFR) and p75(NTR) specific NGF receptors and their contribution in the complex network of tissue repair process, is discussed and highlighted in view of recent findings. In fact, NGF represents a significant advance in the treatment of etiologically different ulcers (corneal ulcers, pressure ulcers, post-viral infections, chemical burns) and might shorten the recovery process. For these diseases, no specific treatment is actually available. It is reasonable that apart from NGF and/or neurotrophins a different time-course of trkA(NGRF)/p75(NTR) expression, might regulate the final process. In summary, these novel findings on the potential pro-healing capacity of NGF might open new possibilities for this growth factor in modulating the healing processes in several pathological conditions.

Cobalamin deficiency-induced down-regulation of p75-immunoreactive cell levels in rat central nervous system.

We investigated immunoreactivity for p75 neurotrophin receptor (NTR) in the spinal cord white matter and septum of rats made cobalamin-deficient (Cbl-D) by means of total gastrectomy or a Cbl-D diet. Cbl deficiency down-regulates p75NTR-immunoreactive cell levels in spinal cord white matter and septum with different time courses. On the whole, the spinal cord white matter seems to be more affected in terms of p75NTR-immunoreactive cells, most of which are astrocytes. The p75NTR-immunoreactive cell levels in the spinal cord white matter and septum normalized in rats treated with Cbl (scheme b) and killed 4 months after total gastrectomy. However, Western blot analysis of p75NTR in the spinal cords of Cbl-D rats shows increased p75NTR protein levels, which are resistant to Cbl replacement. These findings demonstrate that a neurotrophic vitamin (Cbl) positively regulates the levels of a neurotrophic receptor (p75NTR) (at least in terms of immunohistochemistry) in rat central nervous system, although the underlying mechanism(s) are still unknown.

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.

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.

Immunology of the human nail apparatus: the nail matrix is a site of relative immune privilege.

The nail apparatus is constantly exposed to environmental damage. It requires effective immune responses to combat infection, while avoiding the loss of nail production and regeneration by autoaggressive immunity. By immunohistology, we define here previously unknown characteristics of the normal human nail immune system (NIS). Compared with other regions of nail epithelium, human leukocyte antigen (HLA)-A/B/C expression is prominently down regulated on both keratinocytes and melanocytes of the proximal nail matrix (PNM), whereas HLA-G(+) is upregulated here. Together with the expression of macrophage migration inhibitory factor in PNM, this may serve to inhibit an natural killer (NK) cell attack on major histocompatibility complex (MHC) class Ia-negative PNM. PNM also displays strong immunoreactivity for potent, locally generated immunosuppressants such as transforming growth factor-beta1, alpha-melanocyte stimulating hormone, insulin-like growth factor-1, and adrenocorticotropic hormone, exhibits unusually few CD1a(+), CD4(+), or CD8(+), NK, and mast cells. Finally, MHC class II and CD 209 expression on CD1a(+) cells in and around the PNM is reduced, indicating diminished antigen-presenting capacity. Thus, the NIS strikingly differs from the skin immune system, but shows intriguing similarities to the hair follicle immune system, including the establishment of an area of relative immune privilege in the PNM. This nail immune privilege may offer a relative safeguard against autoimmunity. But, the localized intraepithelial defect of innate and adaptive immunity in the PNM revealed here also may impede effective anti-infection defense.

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.

Toll-like receptors and the eye.

PURPOSE OF REVIEW: This 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 FINDINGS: Current 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. SUMMARY: Recent 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.

Impaired brain development in the rat following prenatal exposure to methylazoxymethanol acetate at gestational day 17 and neurotrophin distribution.

Several neuropsychiatric disorders, including schizophrenia, are the consequence of a disrupted development of the CNS. Accordingly, intrauterine exposure to toxins may increase the risk for psychopathology. We investigated whether prenatal exposure of rats to the neurotoxin methylazoxymethanol acetate led to long-term changes in cerebral neurotrophin levels. We measured the brain levels of nerve growth factor and brain derived neurotrophic factor in young adult and adult rats. Decreased nerve growth factor or brain derived neurotrophic factor were found in the parietal cortex accompanied by altered neurotrophin content in the hippocampus and entorhinal cortex. The present study is the first to show long-lasting effects of a single prenatal exposure to a neurotoxin on adult levels of neurotrophins in brain regions implicated in neuropsychiatric disorders.

Altered levels of nerve growth factor in the thymus of subjects with myasthenia gravis.

We have previously reported that nerve growth factor (NGF), a polypeptide known for its neurotrophic activities, is also involved in the differentiation and survival of immune cells, and that NGF and its high-affinity receptor are present in the thymus. We here demonstrate that the thymus of humans affected by myasthenia gravis (MG) contains significant concentrations of NGF. These observations support our hypothesis of a role for NGF in the thymus and suggest that the changes observed in the thymus of subject with MG may have functional significance.

[Nerve growth factor and brain-derived neurotrophic factor: a possible etiopathogenic role in sensorineural hearing loss. Preliminary data]. / Nerve growth factor e brain derived neurotrophic factor: un possibile ruolo eziopatogenico nell'ipoacusia neurosensoriale. Dati preliminari.

Nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) are members of a family of structurally related secreted proteins, termed neurotrophins, which promote and regulate the survival of many kinds of neurons in the peripheral nervous system. Aim of the study is to dose the serum level of NGF and BDNF in 8 patients affected by sensorineural hearing loss (SNHL). The results show that in these patients the amount of circulating NGF, but not of BDNF, is significantly lower in comparison to that found in age-matched controls. The preliminary data of the present study suggest that NGF might have a crucial role in the auditory pathway, promoting the survival and preventing the degeneration of sensorineural cells.