Mb Reichel

Absence of Mutations of the BRAF Gene and Constitutive Activation of Extracellular-Regulated Kinase in Malignant Melanomas of the Uvea

The v-raf murine sarcoma viral homolog B1 (BRAF) gene, one of the human isoforms of RAF, is activated by Ras, leading to cooperative effects in cells responsive to growth factor signals. Recently, somatic missense mutations of the BRAF gene have been detected in more than 66% of malignant melanomas of the skin. We analyzed 42 malignant melanomas of the uvea, 3 corresponding liver metastases, and 10 cutaneous melanomas for possible BRAF mutations: after microdissection, mutation analysis of BRAF and KRAS was performed. The expression of extracellular-regulated kinase 1 and 2 (ERK1/2), an important downstream point of convergence in the Ras-RAF-MEK-Erk pathway, was analyzed immunohistochemically. Interestingly, we failed to detect activating BRAF mutations in uvea melanomas and their corresponding liver metastases. There were no mutations of BRAF in corresponding non-neoplastic uvea specimens, although we detected three BRAF mutations in sporadic cutaneous melanoma that led to a substitution of valine by glutamic acid at position 599 (V599E). KRAS mutations were detected in 1 of 10 cutaneous melanoma but not in uveal or metastatic melanoma. Despite the lack of activating mutations in the BRAF gene, we identified constitutively activated ERK in almost all (86%) uveal melanoma tissues tested but not in corresponding normal retina or uveal cells. Our data indicate that BRAF gene mutations are rare to absent events in uveal melanoma. The finding of activated Erk suggests a causative role for MAPK activation in uveal melanoma independent of activating BRAF or RAS mutations.

High frequency of persistent hyperplastic primary vitreous and cataracts in p53-deficient mice

In order to investigate whether the p53 gene product plays a role in normal eye development, age matched p53-deficient mice and wild-type controls were sacrificed from day 2 to day 21 after birth. Eyes were paraffin-embedded and sectioned. Serial sections were taken at the level of the tunica vasculosa lentis and the hyaloid artery. The terminal dUTP nick-end labelling technique (TUNEL) was used to detect the number of cells displaying DNA fragmentation within these structures. Eyes were also prepared for scanning electron microscopy and resin embedded for semi-thin sections. Adult wild-type mice and p53-deficient mice were examined ophthalmoscopically in vivo. Ophthalmoscopical examination of mice completely deficient in p53 revealed them to be normal except for the persistence of the hyaloid vasculature, a structure that normally regresses during eye development. In adult animals there was also a high frequency of cataracts. Using morphological assessment and TUNEL we could show that in normal mice, regression of the primary vitreous, which includes the hyaloid artery, the vasa hyaloidea propria as well as the tunica vasculosa lentis, occurs via apoptotic cell death within 5 – 6 weeks after birth. The number of TUNEL-positive cells within these structures was significantly reduced in the p53-deficient mice in which parts of the hyaloid vasculature persisted and developed into a fibro-vascular retrolental plaque analogous to persistent hyperplastic primary vitreous (PHPV) described in humans. As in humans, PHPV in mice resulted in the development of cataracts. We have identified a role for p53-dependent apoptosis in the regression of the hyaloid vasculature and tunica vasculosa lentis. Our results provide further evidence for the importance of p53 in normal development and provide the first detailed evidence of its role in postnatal development in remodelling the developing eye.

Gene therapy for inherited retinal degeneration.

Research into the molecular and genetic basis of disease is continually expanding and improving the prospects for rational treatments. Of these, gene therapy (here defined as the introduction of genetic material into human cells) may ultimately offer the greatest scope.1 This article provides a perspective on the potential for gene therapy for treating inherited retinal degenerations, along with an outline of progress and problems encountered to date. Of the inherited forms of retinal degeneration, retinitis pigmentosa (RP) is the best characterised (see Bird2 for review). Seven different non-syndromic RP genes have been identified to date, five of which3-7 are expressed exclusively in photoreceptor cells. However, photoreceptors and retinal pigment epithelial (RPE) cells are in close proximity and are interdependent. Defects in genes expressed in the RPE may also result in retinal degeneration. Delivery to these tissues of either normal copies of the defective genes or genes which enhance cell survival may arrest the degenerative process and thus preserve vision. ### THE EYE AS TARGET ORGAN FOR GENE DELIVERY The eye has a number of advantages as a target organ for gene delivery. It is easily accessible and the tissues may be examined in vivo by ophthalmoscopy. In addition, there are blood-retinal and blood-aqueous barriers which may concentrate vectors in the target area and reduce their spread out of the eye. The eye may be used for testing gene delivery to a wide range of tissues since it contains endothelium (cornea), epithelium (cornea, ciliary body, iris), muscle (ciliary body), and neurons (retina). It may also serve as a valuable model system to test gene therapy strategies for the brain, whose neurons are more difficult to target than those in the neuroretina. ### SCOPE OF GENE THERAPY TO TREAT OCULAR DISORDERS It is probable that gene therapy for certain ocular diseases will be realised sooner than others. Gene therapy strategies for cancers (for example, uveal melanoma), infections …

Co-injection of adenovirus expressing CTLA4-Ig prolongs adenovirally mediated lacZ reporter gene expression in the mouse retina

There is growing interest in gene delivery to the eye in order to develop gene therapy for the many ocular disorders which may be amenable to this approach. To date, recombinant adenoviruses (AV) have been the main vector used for gene delivery to anterior and posterior segments in animal models. As with delivery to other organs, immune responses to vector and transgene limit the duration of expression in the eye. Using an E1-deleted adenoviral vector carrying a lacZ reporter gene, we have previously demonstrated that a T cell-mediated immune response reduces the level of intra-ocular transgene expression over time and limits it to around 3 weeks in mice. This report describes a strategy for prolonging gene expression by blocking the B7–CD28 interactions between antigen presenting cells (APC) and T cells in order to prevent the costimulatory signals required for T cell survival and proliferation. This was achieved by the co-injection of AV encoding a secreted immunomodulatory molecule (CTLA4-Ig) which consists of the extra-cellular domain of mouse CTLA4 fused to the Fc region of human IgG. Subretinal co-injection of AV encoding β galactosidase with AV encoding CTLA4-Ig results in prolonged expression in retinal cells compared with subretinal injection of only adenovirus encoding β galactosidase.

New model of conjunctival scarring in the mouse eye

AIMS To establish a simple model of conjunctival wound healing in the mouse eye. METHODS 4 week old BALB/c mouse eyes were studied over a 14 day period. Surgical procedure under general anaesthesia involved a blunt dissection of the conjunctiva performed by injection of 25 μl of PBS via a 27 gauge needle into one eye, while the contralateral eye was used as control. Mice were assessed clinically and sacrificed at 1, 2, 3, 7, and 14 days after surgery. Enucleated eyes were prepared for histological analysis. Development of scar tissue was studied with haematoxylin and eosin, oxidation aldehyde fuchsin, and van Gieson stains, with assessment of cellularity, extracellular matrix formation, and wound characterisation. RESULTS Histological analysis revealed a marked and characteristic healing response initiated by a predominantly granulocytic inflammatory reaction at day 1 with peak fibroblast activity 3 days after surgery. Oxytalan fibres and newly laid collagen fibres were detected early in the subconjunctival wound area and up to 7 days after surgery. Remodelling and complete organisation of scar tissue was evident by day 14. CONCLUSION A single subconjunctival injection in the mouse eye results in a marked and consistent healing response. This represents a simple, inexpensive, and reliable animal model of conjunctival scarring. The mouse is a biologically well characterised animal model and allows the use of a wide variety of molecular tools. There are potentially significant clinical applications, in particular in investigating the effects of modulating agents such as antimetabolites, growth factors, and their antagonists on conjunctival scarring.

Phenotype of a British North Carolina macular dystrophy family linked to chromosome 6q

AIMS To document the phenotype of an autosomal dominant macular dystrophy diagnosed as having North Carolina macular dystrophy (NCMD) in this British family, and to verify that the disease locus corresponds with that of MCDR1 on chromosome 6q. METHODS 37 family members were examined and the phenotype characterised. DNA samples from the affected members, 19 unaffected and five spouses, were used to perform linkage analysis with six microsatellite marker loci situated within the MCDR1 region of chromosome 6q. RESULTS Every affected family member had lesions characteristic of NCMD, which developed early in life and usually remain stable thereafter. Although fundus changes are evident in the periphery, all tests revealed that functional loss is restricted to the macula. Some patients with large macular lesions had good visual acuity with fixation at the edge of the lesion at 5° eccentricity. Significant linkage to the MCDR1 locus on chromosome 6q was obtained with three marker loci, with a maximum lod score of 5.9 (q = 0.00) obtained with D6S249. CONCLUSION This family has the typical phenotype NCMD, and the causative gene was linked to the disease locus (MCDR1) on chromosome 6q. Early onset and localisation of the disease to the central macula allow specialisation of eccentric retina in some eyes with resultant good visual acuity.

An immune response after intraocular administration of an adenoviral vector containing a beta galactosidase reporter gene slows retinal degeneration in the rd mouse

BACKGROUND/AIMS Retinal degenerations are a leading cause of blindness for which there are currently no effective treatments. This has stimulated interest in the investigation of gene therapy strategies for these diseases in a variety of animal models. A number of attempts have been made to prevent photoreceptor loss in the rd mouse model of retinal degeneration using adenoviral vectors containing either a copy of the missing functional gene or a gene encoding either a neurotrophic factor or an antiapoptotic factor. The authors have previously demonstrated that intraocular administration of an adenoviral vector containing a β galactosidase gene (AV.LacZ) results in an immune response to viral gene products and β galactosidase. Here the effect of the immune response on retinal degeneration is examined. METHODS Juvenilerd mice were injected intravitreally with AV.LacZ and a proportion were depleted of either CD4+ or CD8+ T cells or both. Control animals were injected with PBS. The mice were sacrificed 10 and 20 days post-injection and their eyes embedded in paraffin wax and sectioned. RESULTS 10 days after intravitreal injection of AV.LacZ, the outer nuclear layer contains an average of 2.5 rows compared with 1.5 in PBS injected animals (p<0.005). The protective effect of AV.LacZ is negated by immune suppression and does not extend beyond 20 days. CONCLUSION An immune response to vector and transgene products is able to slow degeneration in the rd mouse. This phenomenon should be taken into account when analysing the degeneration in therd mouse following gene transfer.

Gene transfer in ophthalmology

Background: Research into the molecular and genetic basis of disease is continually expanding. How does the increasing knowledge about the genetic basis of eye diseases contribute to the development of new therapeutic strategies? Materials and methods: Gene therapy, here defined as the introduction of genetic material into human cells, offers great opportunities. Gene transfer strategies can be used for gene replacement in recessive disease, gene inactivation in dominant disease, expression of “rescue factors” and apoptosis modulators in degenerative disease, “suicide genes” for example in proliferative diseases and expression of immunmodulatory factors in immunological disorders. Viral vector systems have been developed to introduce the gene of interest into the target cell. Results: Most of the published strategies include the use of vectors for gene transfer. Adenovirus (AV), adenoassociated virus (AAV), encapsulated adenovirus mini-chromosomes (EAMs), herpes simplex virus (HSV) and lentiviruses are the most frequently used viral vector systems to date. Their advantages and disadvantages, the in vivo models used for gene transfer in retinal degeneration, and the results obtained to date by different research groups in the field will be reviewed. Conclusions: Gene transfer into ocular tissues has been demonstrated with growing functional success and may develop into a new therapeutic tool for clinical ophthalmology.Fragestellung: Die ständig wachsenden Erkenntnisse über die molekularen Ursachen vieler Augenkrankheiten eröffnen auch neue Therapiemöglichkeiten. Wie kann die fortschreitende Genotyp-Phänotyp-Korrelation für die Entwicklung einer Gentherapie genutzt werden? Material und Methoden: Der Gentransfer kann bei rezessiven Erkrankungen für den Genersatz, bei dominanten Erkrankungen für die gezielte Inaktivierung, bei degenerativen Erkrankungen für die Expression von sogenannten „rescue factors“ oder zur Apoptosismodulation, bei proliferativen Erkrankungen für die Expression von „suicide genes“ und bei immunologischen Erkrankungen zur Immunmodulation genutzt werden. Allen Strategien ist die Übertragung von Fremd-DNA in ein Zielgewebe des Auges durch ein Vektorsystem gemeinsam. Ergebnisse: Vor allem virale Vektorsysteme sind bereits für den Gentransfer am Auge untersucht worden: Adenovirus (AV), Adeno-assoziiertes Virus (AAV), Encapsidated adenovirus minichromosomes (EAMs), Herpes-simplex-Virus (HSV) und Lentiviren. Für den Gentransfer in die Netzhaut hat AAV zur Zeit das größte Potential. Die Transduktionseffizienz der Vektorsysteme, ihre Vor- und Nachteile, die verwendeten Tiermodelle und der Stand der Forschung werden diskutiert. Schlußfolgerung: Der Gentransfer ist auch in der Augenheilkunde ein vielversprechender aber immer noch experimenteller Therapieansatz.

Effect of Steroidal and Non-Steroidal Drugs on the Microglia Activation Pattern and the Course of Degeneration in the Retinal Degeneration Slow Mouse

Background: In hereditary retinal degeneration, microglia cells become activated, migrate through the outer nuclear layer (ONL) and accumulate in the subretinal space. Although this inflammatory process is not likely to be responsible for the onset of photoreceptor apoptosis, cytotoxic substances secreted by activated microglia could potentially accelerate and perpetuate the degenerative process. Anti-inflammatory drugs have been shown to modulate the microglia response in neurodegenerative disorders and potentially ameliorate the disease progression in various animal model systems. In this study we wanted to test the impact of the most commonly used anti-inflammatory drugs (acetylsalicylate and prednisolone) on the microglia activation pattern, the rate of caspase-3-dependent photoreceptor apoptosis and the course of the degeneration in the retinal degeneration slow (rds) mouse retina. Methods: 169 pigmented rds mice and 30 CBA wild-type mice were used for this study. The treatment groups were injected daily with either acetylsalicylate (200 mg/kg) or prednisolone (2 mg/kg) i.p. from day 0 up to 3 months. Animals were sacrificed at days 10, 14, 16, 18, 20, 30, 40, 60 and 90. Cryoprotected frozen sections were immunostained with F4/80 and cleaved caspase-3 antibodies. The main outcome measures were the total microglia count in the subretinal space, the total cleaved caspase-3-positive cells in the ONL and the averaged number of photoreceptor rows in the midperipheral retina. Results: Neither acetylsalicylate nor prednisolone reduced subretinal microglia accumulation in the rds mouse degeneration model. Moreover, they aggravated migration and accumulation in the early time course. The apoptotic cascade started earlier and was more pronounced in both treatment groups compared to the control group. The pace of retinal degeneration was not reduced in the treatment groups compared to the untreated control. In contrast, acetylsalicylate did significantly accelerate the photoreceptor cell degeneration in comparison to the prednisolone (p < 0.001) and to the control group (p < 0.001). Conclusions: Acetylsalicylate and prednisolone do not decrease the microglia response in the rds mouse and are not neuroprotective. More research is needed to clarify the molecular mechanisms which lead to photoreceptor cell death and to elucidate the complex role of microglia in inherited retinal degeneration.

Wohin f-hrt die Genrevolution in der Augenheilkunde?

Wenn man bedenkt, dass seit der ersten Linkageanalyse für ein RetinitisPigmentosa-Gen durch S. Bhattacharya nur 18 Jahre vergangen sind, so wird deutlich, wie schnell die Forschung auf diesem Gebiet neue Einblicke in die Pathogenese und damit auch neue Möglichkeiten der Therapie eröffnet hat. Dies gilt insbesondere für die klinisch bedeutendsten und wissenschaftlich am besten untersuchten Degenerationen der äußeren Netzhaut.