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.

Clinical findings in macular hole surgery with indocyanine green-assisted peeling of the internal limiting membrane.

PURPOSE: Indocyanine green (ICG) staining of the internal limiting membrane has facilitated ILM peeling in macular hole surgery. However, it has been reported that ICG-assisted peeling of the ILM may result in retinal damage and unfavorable functional outcome. Therefore, we analyzed our visual and anatomical results of ICG assisted macular hole surgery. METHODS: In a retrospective study the records of a consecutive series of 37 patients with full-thickness idiopathic macular holes operated with ICG-assisted ILM peeling by a single surgeon were analyzed. All patients underwent a standard three-port vitrectomy with surgically induced posterior vitreous detachment, staining of the ILM with ICG, peeling of the ILM in a circular manner around the fovea, and SF6 20% endotamponade. RESULTS: A total of 37 patients (37 eyes) were included in the study. The mean age was 69+/-7 years (range 52-81 years), and there were 26 women and 11 men. The follow-up ranged from 6 to 30 months (mean 18+/-6 months). At baseline visual acuity ranged from 20/400 to 20/40. Anatomically, 13 eyes had stage 2 holes, 21 eyes (57%) stage 3 holes, and three eyes stage 4 holes. At the postoperative visit (8-12 weeks after surgery) anatomical closure of the macular hole was achieved in 36 eyes. Visual acuity ranged between 20/400 and 20/20. At the last follow-up after initial surgery the macular hole was closed in all eyes. Visual acuity ranged from 20/200 to 20/20. CONCLUSION: In our retrospective series anatomical and functional results of macular hole surgery with ICG-assisted peeling of the ILM are satisfactory. Primary hole closure was achieved in 97% of eyes and visual acuity increased in 62% of eyes in our series.

[Progress in somatic gene therapy of retinal degeneration in the animal model]. / Fortschritte in der somatischen Gentherapie von Netzhautdegenerationen am Tiermodell.

More than 60 genes responsible for human retinal dystrophies have already been identified. Most of them are either expressed in the photoreceptor or in the retinal pigment epithelium (RPE). Therefore these cells have become the target of new therapeutical strategies on a molecular level. The most promising approach at present is somatic gene therapy, which has been developed over the last years and the principle has now been established in animal models. For gene therapy of inherited retinal degeneration, as for gene therapy in general, gene transfer has to be proven to be not only efficient but also safe. This has recently been achieved using the adeno-associated virus (AAV) as a vector to express a therapeutic gene within the photoreceptor cell. It could be demonstrated in mouse and dog models of retinal degeneration that expression of the therapeutic transgene leads to anatomical and functional restitution of degenerating photoreceptors. A significant immune response to AAV has not been detected so far. In this paper the recent success of gene therapy of retinal degeneration in animal models is reviewed.

Electrophysiological alterations and upregulation of ATP receptors in retinal glial Müller cells from rats infected with the Borna disease virus.

Infection with the neurotropic Borna disease virus (BDV) causes an immune-mediated neurological disease in a broad range of species. In addition to encephalitis, BDV-infected Lewis rats develop a retinitis histologically characterized by the loss of most retinal neurons. By contrast, the dominating retinal macroglia, the Müller cells, do not degenerate. It is known from several models of neurodegeneration that glial cells may survive but undergo significant alterations of their physiological parameters. This prompted us to study the electrophysiology and ATP-induced changes of intracellular Ca(2+)-concentration ([Ca(2+)](i)) in Müller cells from BDV-infected rat retinae. Freshly isolated cells were used for whole-cell patch-clamp recordings. Whereas neither zero current potentials nor membrane resistances showed significant alterations, the membrane capacitance increased in cells from BDV-infected rats during survival times of up to 8 months. This process was accompanied by a decrease in K(+) current densities. Müller cells from BDV-infected rats were characterized by expression of a prominent fast-inactivating A-type K(+) current which was rarely found in control cells. Moreover, the number of cells displaying Na(+) currents was slightly increased after BDV-infection. ATP evoked increases in [Ca(2+)](i) in Müller cells within retinal wholemounts of both control and BDV-infected animals. However, the number of ATP-responding isolated cells increased from 24% (age-matched controls) to 78% (cells from animals > or =18 weeks after infection). We conclude that in BDV-induced retinopathy, reactive rat Müller cells change their physiological parameters but these changes are different from those in Müller cells during proliferative vitreoretinopathy in man and rabbit.

[Retinal degeneration. Apoptosis as pathomechanism and therapy strategy]. / Netzhautdegenerationen. Apoptose als Pathomechanismus und Therapieansatz.

Molecular techniques in ophthalmology and related subjects have led in recent years to the identification of many genes expressed in photoreceptor cells and have allowed the characterization of mutations leading to distinct phenotypes of retinal degeneration. Programmed cell death, or apoptosis, has been identified as the final common pathway in this disease group. A cascade of events has evolved, starting with specific stimuli and developing over different mediators and regulators (e.g., Fas ligand, proteins of the Bcl-2 family, p53) to effector enzymes (caspases). The ever increasing data of this pathway serve as a basis for new therapeutic strategies. We review the current knowledge on apoptosis in retinal degeneration.

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 beta galactosidase gene (AV.LacZ) results in an immune response to viral gene products and beta galactosidase. Here the effect of the immune response on retinal degeneration is examined. METHODS: Juvenile rd 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 the rd mouse following gene transfer.

Restoration of photoreceptor ultrastructure and function in retinal degeneration slow mice by gene therapy.

The gene Prph2 encodes a photoreceptor-specific membrane glycoprotein, peripherin-2 (also known as peripherin/rds), which is inserted into the rims of photoreceptor outer segment discs in a complex with rom-1 (ref. 2). The complex is necessary for the stabilization of the discs, which are renewed constantly throughout life, and which contain the visual pigments necessary for photon capture. Mutations in Prph2 have been shown to result in a variety of photoreceptor dystrophies, including autosomal dominant retinitis pigmentosa and macular dystrophy. A common feature of these diseases is the loss of photoreceptor function, also seen in the retinal degeneration slow (rds or Prph2 Rd2/Rd2) mouse, which is homozygous for a null mutation in Prph2. It is characterized by a complete failure to develop photoreceptor discs and outer segments, downregulation of rhodopsin and apoptotic loss of photoreceptor cells. The electroretinograms (ERGs) of Prph2Rd2/Rd2 mice have greatly diminished a-wave and b-wave amplitudes, which decline to virtually undetectable concentrations by two months. Subretinal injection of recombinant adeno-associated virus (AAV) encoding a Prph2 transgene results in stable generation of outer segment structures and formation of new stacks of discs containing both perpherin-2 and rhodopsin, which in many cases are morphologically similar to normal outer segments. Moreover, the re-establishment of the structural integrity of the photoreceptor layer also results in electrophysiological correction. These studies demonstrate for the first time that a complex ultrastructural cell defect can be corrected both morphologically and functionally by in vivo gene transfer.

[Gene transfer in ophthalmology]. / Gentransfer in der Augenheilkunde.

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.

[Differential diagnosis of occult primary malignant melanoma]. / Differentialdiagnostische Aspekte von Metastasen maligner Melanome bei unbekanntem Primärtumor.

Controversy exists in literature about therapy and prognosis of malignant melanoma of unknown primary site. We investigated frequency, differential diagnosis and follow-up of patients with occult primary malignant melanoma treated at the University of Leipzig in 1996. Among 135 patients with malignant melanoma (MM) seven were found without known primary. In two of seven cases the medical history pointed to regression of primary lesion of skin. In another two cases the diagnosis "melanoma" was changed to "lung cancer" after autopsy and in one case there was a relationship to a naevus blue resected nine months before. Recurrences or metastases occurred within six months after therapy. Two patients are still alive free of disease after a follow up of 33 and 24 months. Five patients died from tumor progression between 2 to 14 months postoperatively. Pitfalls in differential diagnosis and ways to find out the primary are discussed. Patients with unknown primary malignant melanoma should be treated similar to those with known primary. Radical surgery is indicated because it's impossible to determine the prognosis of patients with unknown primary malignant melanoma.

Transforming growth factor-beta1, -beta2, and -beta3 in vivo: effects on normal and mitomycin C-modulated conjunctival scarring.

PURPOSE: To compare the effects of the three human isoforms of transforming growth factor (TGF)-beta in vivo using a mouse model of conjunctival scarring, both in normal eyes and after treatment with MMC, with a view to delineating the role of this growth factor in glaucoma filtration surgery. METHODS: Application of recombinant human TGF-beta was assessed in a prospective, randomized study of mouse conjunctival scarring, in which subconjunctival TGF-beta1, -beta2, and -beta3 (all 10(-9) M) were compared with control (phosphate-buffered saline [PBS] carrier) and mitomycin C (MMC; 0.4 mg/ml) treatment at 6 hours, and 1, 3, and 7 days after surgery (six eyes/treatment/time point). Effects of TGF-beta2 on eyes previously treated with MMC were also assessed. Histologic studies of enucleated eyes were performed to analyze development of the scarring response, extracellular matrix deposition, and the inflammatory cell profile. RESULTS: All three isoforms of TGF-beta behaved in a similar manner in vivo, being associated with a rapid-onset and exaggerated scarring response compared with control and MMC treatment. TGF-beta-treated eyes showed evidence of an earlier peak in inflammatory cell activity (P < 0.05) and increased collagen type III deposition (P < 0.05). TGF-beta2 treatment significantly stimulated scarring after MMC application (P < 0.05). CONCLUSIONS: TGF-beta1, -beta2, and -beta3 appear to have similar actions in vivo and stimulate the conjunctival scarring response. Application of TGF-beta2 modified the effects of MMC. All TGF-beta isoforms may be potent modulators of the conjunctival scarring response. These studies indicate that TGF-beta2 may naturally modify the antiscarring effects of antimetabolites such as MMC in glaucoma filtration surgery.

Absence of p53 delays apoptotic photoreceptor cell death in the rds mouse.

PURPOSE: This study was aimed at determining whether or not apoptotic photoreceptor cell death in a mouse model of inherited retinal degeneration is p53 dependent. METHODS: A colony of p53-deficient rds mice were obtained by crossing homozygous rds mice with animals homozygous for a targeted disruption of the p53 gene and genotyping the offspring of the F1 cross. Both parental strains were on a BALB/c background. Age matched p53-deficient rds mice and controls (p53-deficient, rds and BALB/c mice), were sacrificed from day 1 to day 58 after birth. Eyes were paraffin-embedded and a modified terminal dUTP nick-end labeling (TUNEL) technique was used to detect the number of cells displaying DNA fragmentation within the sectioned retina. Eyes were also resin-embedded for semi-thin and ultra-thin sectioning. RESULTS: The peak in photoreceptor apoptosis, which occurs at 16 days in the rds mouse, was delayed by 3 days in p53-deficient rds mice. In addition, there was also a delay in the loss of photoreceptor cells between 16 and 26 days. However, absence of p53 did not prevent retinal degeneration in the rds mouse. The number of photoreceptor cells in p53-deficient rds mice at 35 days was very similar to that in the controls. CONCLUSIONS: We have demonstrated that absence of p53 delays but does not prevent photoreceptor cell loss in the rds mouse. Our results provide evidence for plasticity in the mechanism by which apoptosis proceeds in retinal degeneration.

Adeno-associated virus gene transfer to mouse retina.

Ocular gene transfer may provide a means for arresting the retinal degeneration characteristic of many inherited causes of blindness, including retinitis pigmentosa (RP). Previously, we have shown in immunodeficient animals that recombinant adeno-associated virus (rAAV) mediates transduction of photoreceptors as well as the retinal pigment epithelium (RPE) following subretinal injection. In this study we extend these observations and show that highly purified recombinant AAV vectors encoding the reporter gene LacZ transduce photoreceptors in an immunocompetent mouse strain following subretinal injection and efficiently transduce ganglion cells after intravitreal injection. Levels of transduction increase over time. Sublethal gamma-irradiation is shown to facilitate this process.

Characterization of the regulatory regions in the human desmoglein genes encoding the pemphigus foliaceous and pemphigus vulgaris antigens.

The adhesive proteins in the desmosome type of cell junction consist of two members of the cadherin superfamily, the desmogleins and desmocollins. Both desmogleins and desmocollins occur as at least three different isoforms with various patterns of expression. The molecular mechanisms controlling the differential expression of the desmosomal cadherin isoforms are not yet known. We have begun an investigation of desmoglein gene expression by cloning and analysing the promoters of the human genes coding for the type 1 and type 3 desmogleins (DSG1 and DSG3). The type 1 isoform is restricted to the suprabasal layers of the epidermis and is the autoantigen in the autoimmune blistering skin disease pemphigus foliaceous. The type 3 desmoglein isoform is also expressed in the epidermis, but in lower layers than the type 1 isoform, and is the autoantigen in pemphigus vulgaris. Phage lambda genomic clones were obtained containing 4.2 kb upstream of the translation start site of DSG1 and 517 bp upstream of the DSG3 start site. Sequencing of 660 bp upstream of DSG1 and 517 bp upstream of DSG3 revealed that there was no obvious TATA box, but a possible CAAT box was present at -238 in DSG1 and at -193 in DSG3 relative to the translation start site. Primer extension analysis and RNase protection experiments revealed four putative transcription initiation sites for DSG1 at positions -163, -151, -148 and -141, and seven closely linked sites for DSG3, the longest being at -140 relative to the translation start site. The sequences at these possible sites at -166 to -159 in DSG1 (TTCAGTCC) and at -124 to -117 in DSG3 (CTTAGACT) have some similarity to the initiator sequence (CTCANTCT) described for a TATA-less promoter often from -3 to +5, and the true transcription initiator site might therefore be the A residue in these sequences. There were two regions of similarity between the DSG1 and DSG3 promoters just upstream of the transcription initiation sites, of 20 and 13 bp, separated by 41 bp in DSG1 and 36 bp in DSG3. The significance of these regions of similarity remains to be elucidated, but the results suggest that they represent a point at which these two desmoglein genes are co-ordinately regulated. Analysis of the upstream sequences revealed GC-rich regions and consensus binding sites for transcription factors including AP-1 and AP-2. Exon boundaries were conserved compared with the classical cadherin E-cadherin, but the equivalent of the second cadherin intron was lacking. A 4.2 kb region of the human DSG1 promoter sequence was linked to the lacZ gene reporter gene in such a way that there was only one translation start site, and this construct was used to generate transgenic mice. We present the first transgenic analysis of a promoter region taken from a desmosomal cadherin gene. Our results suggest that the 4.2 kb upstream region of DSG1 does not contain all the regulatory elements necessary for correct expression of this gene but might have elements that regulate activity during hair growth.

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 BABL/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 microliters 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.

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 beta galactosidase with AV encoding CTLA4-Ig results in prolonged expression in retinal cells compared with subretinal injection of only adenovirus encoding beta galactosidase.

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 degrees 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.

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.

Immune responses limit adenovirally mediated gene expression in the adult mouse eye.

In order to investigate the immunological consequences of gene transfer to the eye using viral vectors, adenovirus carrying a lacZ reporter gene (AV.LacZ) was injected either subretinally, subconjunctivally or into the anterior chamber of three groups of adult mice: immunocompetent or transiently immunosuppressed BALB/c mice and congenic immunodeficient nude mice. Adenovirally mediated lacZ expression persisted for approximately 3 weeks following injection of the vector into the anterior chamber, retina or extra ocular tissues of the conjuctiva of BALB/c mice. It appears that T cell-mediated immune responses limit the duration of AV-mediated ocular gene expression in adult mice since lacZ gene expression was detected for at least 15 weeks in T cell-deficient BALB/c nude mice, although the level of transgene expression decreased with time. Since intra-ocular AV-mediated gene expression was not significantly longer than extra-ocular expression, it appears that the eye is not normally immune-privileged with respect to viral vectors. Inflammatory cells were detected in the vitreous after anterior chamber injection and in the retina after subretinal injection of adenovirus. The presence of both CD4+ and CD8+ T cells was established by immunophenotyping. Reinjection of BALB/c mice resulted in rapid decline in reporter gene expression, but successful readministration was possible in the case of immunodeficient nude mice. However, after transient depletion of T cells, achieved by intraperitoneal injection of both CD8- and CD4-specific antibodies, the duration of expression in BALB/c mice was longer in the eye (at least 12 weeks, again with decrease in level over time), than in extra-ocular tissues (8 weeks) provided the animal was not reinjected with virus raising the possibility of partial ocular immune-privilege after transient immunosuppression.

Small-cell lung cancer is characterized by a high incidence of deletions on chromosomes 3p, 4q, 5q, 10q, 13q and 17p.

The genetic mechanisms that define the malignant behaviour of small-cell lung cancer (SCLC) are poorly understood. We performed comparative genomic hybridization (CGH) on 22 autoptic SCLCs to screen the tumour genome for genomic imbalances. DNA loss of chromosome 3p was a basic alteration that occurred in all tumours. Additionally, deletions were observed on chromosome 10q in 94% of tumours and on chromosomes 4q, 5q, 13q and 17p in 86% of tumours. DNA loss was confirmed by loss of heterozygosity (LOH) analysis for chromosomes 3p, 5q and 10q. Simultaneous mutations of these six most abundant genetic changes were found in 12 cases. One single tumour carried at least five deletions. DNA under-representations were observed less frequently on chromosome 15q (55%) and chromosome 16q (45%). The prevalent imbalances were clearly indicated by the superposition of the 22 tumours to a CGH superkaryogram. In our view, the high incidence of chromosomal loss is an indication that SCLC is defined by a pattern of deletions and that the inactivation of multiple growth-inhibitory pathways contributes in particular to the aggressive phenotype of that type of tumour.