Treatment of glaucomatous patients by means of food supplement to reduce the ocular discomfort: a double blind randomized trial.

BACKGROUND AND AIM: Chronic use of multi-dose eye drops containing preservatives, such as it may happen in patients affected by primary open angle glaucoma, often results in a damage of the ocular surface due to the inherent toxicity of preservatives, that with time may lead to a lacrimal dysfunction syndrome and eye dryness. PATIENTS AND METHODS: This double blind, randomized, pilot study was conducted on 38 glaucomatous patients suffering from dry eye induced by long-term use of eye drops preserved with BAK. RESULTS: Treatment of these patients with a food supplement containing an association of forskolin, rutin and vitamins B1 and B2 for 30 days increased significantly their OPI values and improved the symptoms of dry eye with respect to a placebo-treated control group. CONCLUSIONS: The association of forskolin, rutin and vitamins B1 and B2 appears to be protective for the ocular surface, contributing to restore a normal equilibrium of the tear film in those subjects in which toxic agents such as BAK had determined alterations of its homeostasis.

Forskolin and rutin prevent intraocular pressure spikes after Nd:YAG laser iridotomy.

AIM: the purpose of this research was to evaluate whether an oral treatment with an association of forskolin and rutin can blunt the intraocular pressure (IOP) spikes and avoid the damage that may occur after laser iridotomy. METHODS: Ten patients underwent bilateral Neodymium:YAG (Nd:YAG) laser iridotomy (Visulas YAG III Laser, Zeiss), for the prevention of primary closed-angle glaucoma. IOP was measured in subjects before and after 7 days of pretreatment with placebo or forskolin and rutin by Goldman applanation tonometry. The IOP was measured before surgery and after surgery at 30-60-120 minutes, and 4-7 days. RESULTS: Analysis of variance indicated a significant increase of the postoperative values in patients receiving treatment with placebo (p < 0.001), but not in those who received treatment with the forskolin and rutin association. T test analysis confirmed that IOP still remained significantly elevated 7 days after laser intervention in placebo treated patients, whereas it stayed within normal values in forskolin/rutin treated patients. CONCLUSION: Forskolin and rutin can blunt the increase of IOP that occurs after Nd-YAG laser iridotomy. This can avoid serious risk to the optic nerve of the patients under laser treatment for iridotomy.

The effect of night vision goggles on the retinocortical bioelectrical activity and its improvement by food supplement.

AIM: To investigate the effect of luminance variations, as well as the oral administration of a food supplement, on the visual bioelectric response while using of Night Vision Goggles (NVG). METHODS: Two trials were performed, both enrolling healthy male aircrew members wearing NVG, and recording Visual Evoked Potentials (VEPs) from scalp electrodes. Both foveal and parafoveal response were evaluated. Latency and amplitude, P100 peak, were measured. In the first set of measurements, VEPs parameters were recorded during unaided photopic conditions and mesopic conditions while using 3rd generation plus NVG (ANVIS 9). In the second set of experiments, after the first basal electrophysiological investigation during mesopic conditions using NVG, patients started a 45 days oral treatment, during which they took 3 tablets per day of a food supplement. The tablets contained a mix of anthocyanosides, procyanidolic oligomers, lutein and vitamins A and E. At the end of this treatment, patients were tested again by pattern-reversal VEP investigation during aided vision condition (wearing NVG) in a mesopic environment. RESULTS: VEPs parameters, statistically evaluated using a two tailed paired t-test, showed that latency and amplitude were respectively increased (p < 0.001 and p < 0.01 for 15' and 60' minutes of arc) and decreased (p < 0.05) when measured using NVG with respect to unaided basal conditions. Furthermore, the VEP response in NVG aided vision was positively affected by the oral treatment with the food supplement, showing a significant (p < 0.05) decrease of latency and increase of amplitude. CONCLUSION: The use of NVG impairs the VEP response, and such effect is effectively counteracted by the oral treatment with a food supplement containing a combination of sight improving molecules that might enhance foveal selectivity, central photoreceptors sensitivity and magnocellular fibers effectiveness.

Retrospective study of glaucoma and closed-eyelid test: long-term outcomes in an Italian native population.

AIM: To establish a threshold value of intraocular pressure (IOP) increase after the closed-eyelid test (CET) that correlates with the highest probability of developing overt primary open-angle glaucoma (OAG) in an Italian native population from 1980 to 2010. METHODS: Retrospective analysis of data obtained from 161 patients with ocular hypertension who performed the CET in 1980, and were subsequently followed to see whether they developed OAG. CET was performed always in the morning Eyelids were closed by bandaging for 1 h in a quiet environment, with the patient seated and not sleeping. IOP was measured again 8 to 10s after opening the eyelids. RESULTS: Accurate statistical analysis of the obtained values indicated that 77% of the subjects showing an IOP increase after 1 hour of eyelid closure in a sitting position developed OAG in the following 30 years and that IOP increase values above 4 mmHg led to a subsequent diagnosis of glaucoma in more than 80% of the patients. CONCLUSION: Eyelid closure for 60 minutes results in a net elevation of IOP the extent of which depends on the balance between the increase of aqueous humour secretion and its outflow. Therefore, the CET may discriminate individuals with a normal outflow from individuals with a less functional outflow, which are evidently those at a higher risk of developing glaucoma.

Cytokeratin expression in primary epithelial cell culture from bovine conjunctiva.

Aim of the present study is to extend our previous observations on a model of primary epithelial cell culture obtained from bovine conjunctiva, and analyse the maintenance of the conjunctival phenotype, relative to cytokeratin (CK) expression, through extended periods of cultivation under different conditions. Conjunctival epithelial cells were grown in transwell filters, and cultured either under liquid covered (LC), or air-interface (AI) conditions. The physiological state of the cells was monitored daily by measurement of the trans-epithelial electrical resistance (TEER). Analysis of cytokeratin expression was then carried out at different time points (up until 14 days), and compared to the original profile of the conjunctival tissue in order to assess deviations from the primitive phenotype. Immunodetection studies, carried out by both western immunoblot and immunofluorescence analyses, revealed constant expression of the pan-epithelial marker AE3 (recognizing basic type cytokeratins), confirming the epithelial nature of the culture. Other cytokeratins characteristic of non-keratinized stratified epithelia (CK4 and CK13) were absent in corneal tissue, while in conjunctival epithelial cells were more expressed under AI than under LC culture conditions. Expression of CK12, a specific marker of corneal tissue, revealed by the antibody AE5, was never observed in conjunctival epithelial cells. These results indicate that the conjunctival phenotype is conserved during extended periods of culturing, making this system a reliable substitute of conjunctival tissue for pharmaceutical analyses.

[Metastatic uveal melanoma. Development of neoadjuvant treatment in animal experiments]. / Metastasierendes uveales Melanom. Tierexperimentelle Entwicklung neoadjuvanter Therapien.

BACKGROUND: Up to 50% of patients with uveal melanoma develop metastases but none of the existing treatments of the primary tumor has been able to reduce the metastatic rate. Probably, micrometatases have already developed before treatment of the uveal melanoma and dormant micrometastases can persist for years before they start growing. This long time-span provides the possibility to treat micrometastases. METHODS: In order to develop an animal model for metastatic uveal melanoma, B16 melanoma cells were injected into the posterior ocular compartment of C57BL6 mice. These cells grew and metastasised to the lungs and liver. Immunological factors for the metastatic process and possible neoadjuvant treatments were investigated. RESULTS: Natural killer cells (NK) are of significance in the rejection of metastases and HLA-I expression of uveal melanomas correlates with the melanoma cell type. Interferon-alpha-2b increases the activity of NK cells and reduces the metastatic rate in the animal model. CONCLUSION: Treatment with interferon-alpha-2b results in decreased metastases from intraocular melanoma in a murine model.

Mechanisms regulating c-met overexpression in liver-metastatic B16-LS9 melanoma cells.

Liver selected B16-LS9 melanoma cells show a dramatic overexpression of the proto-oncogene c-met, the cellular receptor for hepatocyte growth factor/scatter factor. As a consequence, c-met becomes constitutively active, and the cells become more responsive to hepatocyte growth factor stimulation. We have investigated the molecular mechanisms regulating c-met expression in both the parental line B16-F1, which has low expression levels, and the liver-specific B16-LS9, overexpressing c-met. Overexpression is observed at the protein and mRNA levels, however without further evidence of gene amplification or rearrangement. c-met promoter activity was higher in B16-LS9 than B16-F1 cells, and also a nuclear run-off showed higher transcription levels in B16-LS9 cells. Moreover, we found that c-met mRNA had a longer half-life in B16-LS9 cells, thus indicating also the involvement of post-transcriptional regulation mechanisms. Finally, we found evidence that autonomous activation of the melanocortin receptor-1 (MCR-1) is at least partially responsible for c-met upregulation in B16-LS9 cells, since treatment of the cells with a potent MSH antagonist (the agouti peptide) has strong down-regulatory effects.

Differentiation and metastasis in melanoma.

The incidence of melanoma has been rising steadily during the last four decades and is now among the highest of all human cancers. As for most tumors, malignancy and metastatic spreading represent the deadly aspects of a tumor that, if eradicated before becoming invasive, can be easily cured. In fact, melanoma metastatic to regional lymph nodes carries a poor prognosis, and distant metastatic melanoma becomes incurable. Because traditional forms of chemotherapy have little effect on this type of tumor, differentiation therapy has been considered as a possible alternative, based on the consideration that malignancy and differentiation are usually inversely related. However, the relationship between these two factors turned out to be more complex for melanoma cells, and in some murine model systems it has been found that differentiated cells, although less tumorigenic, could be even more metastatic. No clear correlation has been reported between (epi)genetic changes induced by differentiating drugs and the increased malignant phenotype. This review examines what is known to date about differentiation state and metastatic ability of melanomas, and also reports some novel data with the B16 mouse melanoma model system.

Neoadjuvant interferon alfa-2b treatment in a murine model for metastatic ocular melanoma: a preliminary study.

OBJECTIVES: To investigate the treatment of metastasis from uveal melanoma and to test the effect of interferon (IFN) alfa-2b in a murine model. METHODS: The B16-LS9 tissue culture melanoma cells were inoculated into the posterior intraocular compartment of 3 groups of C57BL/6 mice. The inoculated eyes were enucleated at 9 days and the mice were euthanized at 26 days after inoculation; the site and number of metastases were determined using standard histologic techniques. Group 1 was the control group; group 2 was given 20,000 international units (IU) of IFN alfa-2b intramuscularly 12 hours before enucleation, and group 3 received daily injections of 20,000 IU of IFN alfa-2b intramuscularly starting 4 days before enucleation. RESULTS: Pulmonary metastases were detected in 57%, 33%, and 0% of groups 1, 2, and 3, respectively; hepatic micrometastases were detected only in group 1. These results showed a significant decrease in hepatic metastases in mice receiving IFN alfa-2b vs controls (P =.005). CONCLUSION: Treatment with IFN alfa-2b results in decreased hepatic metastases from intraocular melanoma in a murine model. Arch Ophthalmol. 2000;118:1085-1089

A new technique for implantation of tissue culture melanoma cells in a murine model of metastatic ocular melanoma.

The aim of this study was to compare the transcorneal and transconjunctival techniques for the implantation of intraocular melanoma cells and development of metastasis in a murine model. Groups of C57BL/6 mice were given either transconjunctival or transcorneal inoculations of 2.5 x 10(5)/2.5 microl tissue culture B16-LS9 melanoma cells into the intraocular posterior compartment (PC). The eyes were enucleated at 4-11 days post-inoculation and histologically examined. The mice were sacrificed 14 days after enucleation and necropsies were performed with histological evaluation for visceral metastases. Intraocular and extraocular tumour growth was present in all of the eyes inoculated via the transconjunctival route. Pulmonary metastases were found in this group if the eye was enucleated 7 or more days post-inoculation. The melanoma remained confined to the inside of the eye in the transcorneal group until day 7. Haematogenous metastases to the lung and liver developed from the intraocular melanoma in this group. Transcorneal inoculation of tissue culture melanoma cells into the murine PC provides a useful animal model for visceral metastasis of ocular melanoma.

Depletion of NK cell activity results in growth of hepatic micrometastases in a murine ocular melanoma model.

PURPOSE: To study the role of natural killer (NK) cells in growth of spontaneous hepatic metastasis in a murine intraocular melanoma model. METHODS: Tissue culture B16-LS9 melanoma cells were analyzed by flow cytometry for MHC class I expression of all haplotypes and inoculated into the posterior compartment (PC) of one eye of C57BL6 mice. The eyes were enucleated at 12 days post-inoculation and histologically examined for tumor growth. One group of mice (n = 10) were given intraperitoneal injections of anti-asialo GM1 for NK cell depletion post-enucleation and a second group of mice (n = 9) served as controls. The mice were sacrificed at 24 days post-inoculation and necropsies were performed to determine the number and size of metastasis. RESULTS: The B16-LS9 cells failed to express MHC class I antigen. Tumor grew in the PC of all eyes and metastasized to the lungs and livers of all mice, with the average number of hepatic micrometastases greater in the NK depleted group versus the control group (p =.009). There was no significant difference in the average number of pulmonary metastases in the treated versus the control group (p =.072). Hepatic metastases grew to an average diameter of 600 microm in diameter in two NK depleted mice. CONCLUSIONS: NK depletion in this model of metastatic ocular melanoma results in increased number and growth of hepatic micrometastases.

B16LS9 melanoma cells spread to the liver from the murine ocular posterior compartment (PC).

PURPOSE: To create a murine ocular melanoma model that consistently metastasizes to the liver. METHODS: Twelve-week-old C57BL6 mice (n=10) were inoculated in the posterior compartment (PC) of one eye with 5x10(5) tissue culture B16LS9 melanoma cells. The inoculated eyes were enucleated at two weeks and the mice were sacrificed with necropsies performed at four weeks post-inoculation. RESULTS: Melanoma grew and was confined to the eyes of all 10 mice. The melanoma hematogenously spread to the lungs in 9 of 10 mice and the liver in 8 of 10 mice. There was a positive correlation between pulmonary and liver metastasis (r=0.94). CONCLUSIONS: B16LS9 melanoma cells consistently hematogenously spread to the liver when implanted into C57BL6 mice eyes. The value of this model is unknown at this time since it is not known if the intrahepatic melanoma is capable of growing and killing the host.

Regulation of c-met expression in B16 murine melanoma cells by melanocyte stimulating hormone.

B16 murine melanoma cells selected in vivo for enhanced liver metastatic ability (B16-LS9) show on the one hand an increased expression and constitutive activation of the proto-oncogene c-met (the receptor for hepatocyte growth factor/scatter factor), and on the other hand a more differentiated phenotype, when compared to the parental cell line, B16-F1. Following this observation, we have tried to establish whether there is a direct relationship between differentiation and c-met expression in B16 melanoma cells. Treatment of these cells with differentiating agents indicated that c-met expression was strongly induced by melanocyte stimulating hormone, while retinoic acid had almost no influence. c-met induction was triggered by engagement of the melanocortin receptor, cAMP elevation and PKA/PKC(&agr;) activation, as respectively shown by the effects of ACTH, cAMP elevating agents and specific PK inhibitors. Regulation of c-met expression via the melanocortin receptor and cAMP raises the intriguing possibility that autocrine and/or paracrine mechanisms acting in vivo on this circuit might influence (through c-met expression and activation) the metastatic behavior of these tumor cells, which we have shown to be dependent on their c-met expression.

Hepatocyte growth factor/scatter factor and hepatocytes are potent downregulators of tyrosinase expression in B16 melanoma cells.

Reiterated selection in vivo of B16 murine melanoma cells for enhanced liver metastatic ability yielded a cel line (B16-LS9) dramatically overexpressing a constitutively active hepatocyte growth factor/scatter factor (HGF/SF) receptor, the product of the c-met proto-oncogene. Most likely because of their overexpressing c-met, B16-LS9 cells appear to be more responsive than parental B16-F1 cells to HGF stimulation, in terms of motility, invasion, and growth. They are also more pigmented, and express higher levels of tyrosinase as compared to parental B1 6-F1 cells. Therefore, we set out to explore whether HGF/SF and the liver might influence the differentiation state of B1 6 cells. We found that HGF/SF and MSH, two factors which reportedly have a strong influence on the phenotype and the malignant behavior of melanoma cells, may act at different levels, and with opposite results, on the regulation of gene expression. In fact, while MSH induces, at the transcriptional level, an increase in the production of both c-met and tyrosinase, HGF/SF, in contrast, promotes a decrease in the expression of both c-met and tyrosinase, however at a posttranscriptional level. These two opposite effects can counter-balance each other, when the cells are treated with both factors at the same time, apparently through a mechanism involving MAP kinase activation. The effects were, however, additive when morphological changes were considered. Most intriguingly, we also describe a very strong downregulatory activity, limited to tyrosinase expression, by hepatocytes in coculture with B16 cells. This activity, also at the posttranscriptional level, is much stronger than that exerted by HGF/SF, and appears to be due to a labile soluble factor produced by the hepatocytes.

Influence of hepatocyte growth factor/scatter factor on the metastatic phenotype of B16 melanoma cells.

B16 melanoma cells selected in mice for liver-specific metastasis (B16-LS9) overexpress a constitutively active form of the hepatocyte growth factor/scatter factor receptor (HGF/SFr), the product of the c-met proto-oncogene. HGF/SF can affect both invasion and growth of receptive cells. In fact, we show that overexpression of c-met in B16-LS9 cells results in a higher inducibility of two different proteolytic activities (uPA and gelatinase), in correlation with a stronger invasive and motility response to HGF/SF treatment. However, HGF/SF treatment inhibits growth of B16 cells, which might appear in contradiction with the observation that c-met overexpression and constitutive activation seems to be required for efficient liver colonization. However, this apparent discrepancy is resolved by the finding that liver-derived, but not lung-derived factor(s), can efficiently rescue B16-LS9 cells from the growth inhibitory effects of HGF/SF, while not changing their motility response. Therefore, overexpression of c-met in B16-LS9 cells might give a specific advantage in liver colonization, because specifically at this site B16-LS9 cells can take full advantage of the positive effects exerted by HGF/SF stimulation on motility and invasion, while the negative effects on growth are counteracted by other paracrine factor(s).

C-met activation is necessary but not sufficient for liver colonization by B16 murine melanoma cells.

Metastasis to the liver is a frequent event in clinical oncology, the molecular mechanisms of which are not fully understood. We have recently reported a consistent overexpression of c-met in B16 melanoma cells selected in vivo for enhanced liver metastatic ability. In this study we address the question as to whether constitutive activation of c-met is a necessary and sufficient condition for enhanced liver colonization by B16 melanoma cells. Different levels of c-met expression and/or activation in B16 cells were achieved by subcloning, or by c-DNA transfection with either HGF/SF or the oncogenic form of c-met (tpr-met). Metastatic ability of the different populations was then evaluated in vivo by the lung colonization (experimental metastasis) assay. Results indicate that c-met (but not tpr-met) activation in B16 melanoma cells may increase their liver colonizing potential, probably by enhancing motility and invasion in response to paracrine interactions with its ligand. C-met expression per se, however, is not able to change the organ specificity of the cells. C-met activation appears instead to be required at later stages of liver colonization by B16 melanoma cells, in order to enhance their site-specific metastatic ability.

Constitutive activation of c-Met in liver metastatic B16 melanoma cells depends on both substrate adhesion and cell density and is regulated by a cytosolic tyrosine phosphatase activity.

Serial selection in vivo for liver colonization of B16 murine melanoma cells consistently resulted in cell lines expressing elevated amounts of the hepatocyte growth factor/scatter factor receptor (c-Met), which is constitutively activated in the absence of its cognate ligand. In this paper we present evidence suggesting that c-Met constitutive activation in liver-specific B16 melanoma cells depends on both receptor concentration on the cell surface and a cytosolic tyrosine phosphatase activity. In fact, c-Met constitutive activation is suddenly lost upon detachment of the cells from the substrate and is dramatically decreased in adherent cells plated at low density. The loss of tyrosine phosphorylation of c-Met in suspension appears to depend, at least partly, on an increased cytosolic tyrosine phosphatase activity. Instead, lower activation of c-Met at low density mostly results from a decrease in receptor concentration on the membrane. Moreover, we show that c-Met activation does not occur homogeneously on the surface of adherent cells. In fact, receptor concentration and activation appear to be higher on the ventral surface (adherent to the substrate) than on the apical surface. Upon detachment, compartmentalization is lost, leading to a decrease in average receptor density on the plasma membrane and hence to a lower activation.

Expression of constitutively activated hepatocyte growth factor/scatter factor receptor (c-met) in B16 melanoma cells selected for enhanced liver colonization.

The murine melanoma B16-LS9 cell line was obtained after repeated passages in vivo through the liver of syngeneic mice, and shows an enhanced ability to colonize the liver after intravenous inoculation when compared to its parental, unselected counterpart B16-F1. We have previously shown that paracrine growth effects mainly account for better growth of B16-LS9 in the liver than at other sites, and more recently we reported hepatic transferrin as a factor contributing to efficient growth in the liver. Here we show that increased expression of constitutively activated c-met (the receptor for Hepatocyte Growth Factor/Scatter Factor) consistently occurs during selection of B16 cells through the liver. Constitutive activation of c-met seems to follow its own overexpression and not to depend on an autocrine mechanism. As a consequence, liver-selected B16 melanoma cells have higher tyrosine-kinase activity and higher amounts of tyrosine-phosphorylated proteins than parental B16-F1 or lung-specific B16-F10 cells. Overexpression of constitutively activated c-met enhances motility and invasiveness of B16-LS9 cells, presumably favoring their colonization efficiency in vivo. However, whether levels of c-met expression also determine the organ-specificity of B16 melanoma cells needs further clarification.

Murine models of liver metastasis.

Even though the liver is a relevant metastatic site for several human malignant tumors, mechanisms or organ-specific metastasis to the liver remain largely unknown. In the following paper we summarize the results obtained with different murine model systems which have been set up to elucidate the above mechanisms, and describe our own results with two murine models: the F9 teratocarcinoma and the B16 melanoma. While the F9 teratocarcinoma model system underscores the roles of both adhesion and growth stimulation in the target organ, the B16 melanoma model strengthens the relevance of paracrine growth stimulation. Moreover, B16 melanoma cells selected in vivo for increased liver colonization ability appear to depend on cell-to-cell contact with hepatocytes in order to gain efficient growth stimulation. When we next tried to identify the molecule(s) responsible for the growth effect in a liver plasma membrane extract, we found that such activity was mediated by two closely related protein bands. These turned out to be two different forms of transferrin (Tf), one of which is specifically present on the hepatocyte surface. Moreover, when we analyzed the different B16 lines for the expression of c-Met[the receptor for the hepatocyte growth factor-scatter factor (HGF/SF)], we found that liver-specific LS9 had more of this protein than lung-specific F10 or parental F1, suggesting a role for HGF/SF in liver colonization by B16 melanoma cells.