Effects of ciliary muscle plasmid electrotransfer of TNF-alpha soluble receptor variants in experimental uveitis.

Intraocular inflammation has been recognized as a major factor leading to blindness. Because tumor necrosis factor-alpha (TNF-alpha) enhances intraocular cytotoxic events, systemic anti-TNF therapies have been introduced in the treatment of severe intraocular inflammation, but frequent re-injections are needed and are associated with severe side effects. We have devised a local intraocular nonviral gene therapy to deliver effective and sustained anti-TNF therapy in inflamed eyes. In this study, we show that transfection of the ciliary muscle by plasmids encoding for three different variants of the p55 TNF-alpha soluble receptor, using electrotransfer, resulted in sustained intraocular secretion of the encoded proteins, without any detection in the serum. In the eye, even the shorter monomeric variant resulted in efficient neutralization of TNF-alpha in a rat experimental model of endotoxin-induced uveitis, as long as 3 months after transfection. A subsequent downregulation of interleukin (IL)-6 and iNOS and upregulation of IL-10 expression was observed together with a decreased rolling of inflammatory cells in anterior segment vessels and reduced infiltration within the ocular tissues. Our results indicate that using a nonviral gene therapy strategy, the local self-production of monomeric TNF-alpha soluble receptors induces a local immunomodulation enabling the control of intraocular inflammation.

Influence of age on retinochoroidal healing processes after argon photocoagulation in C57bl/6j mice.

PURPOSE: To analyze the influence of age on retinochoroidal wound healing processes and on glial growth factor and cytokine mRNA expression profiles observed after argon laser photocoagulation. METHODS: A cellular and morphometric study was performed that used 44 C57Bl/6J mice: 4-week-old mice (group I, n=8), 6-week-old mice (group II, n=8), 10-12-week-old mice (group III, n=14), and 1-year-old mice (group IV, n=14). All mice in these groups underwent a standard argon laser photocoagulation (50 microm, 400 mW, 0.05 s). Two separated lesions were created in each retina using a slit lamp delivery system. At 1, 3, 7, 14, 60 days, and 4 months after photocoagulation, mice from each of the four groups were sacrificed by carbon dioxide inhalation. Groups III and IV were also studied at 6, 7, and 8 months after photocoagulation. At each time point the enucleated eyes were either mounted in Tissue Tek (OCT), snap frozen and processed for immunohistochemistry or either flat mounted (left eyes of groups III and IV). To determine, by RT-PCR, the time course of glial fibrillary acidic protein (GFAP), vascular endothelial growth factor (VEGF), and monocyte chemotactic protein-1 (MCP-1) gene expression, we delivered ten laser burns (50 microm, 400 mW, 0.05 s) to each retina in 10-12-week-old mice (group III', n=10) and 1-year-old mice (group IV', n=10). Animals from Groups III' and IV' had the same age than those from Groups III and IV, but they received ten laser impacts in each eye and served for the molecular analysis. Mice from Groups III and IV received only two laser impacts per eye and served for the cellular and morphologic study. Retinal and choroidal tissues from these treated mice were collected at 16 h, and 1, 2, 3, and 7 days after photocoagulation. Two mice of each group did not receive photocoagulation and were used as controls. RESULTS: In the cellular and morphologic study, the resultant retinal pigment epithelium interruption expanse was significantly different between the four groups. It was more concise and smaller in the oldest group IV (112.1 microm+/-11.4 versus 219.1 microm+/-12.2 in group III) p<0.0001 between groups III and IV. By contrast, while choroidal neovascularization (CNV) was mild and not readily identifiable in group I, at all time points studied, CNV was more prominent in the (1-year-old mice) Group IV than in the other groups. For instance, up to 14 days after photocoagulation, CNV reaction was statistically larger in group IV than in group III ((p=0.0049 between groups III and IV on slide sections and p<0.0001 between the same groups on flat mounts). Moreover, four months after photocoagulation, the CNV area (on slide sections) was 1,282 microm(2)+/-90 for group III and 2,999 microm(2)+/-115 for group IV (p<0.0001 between groups III and IV). Accordingly, GFAP, VEGF, and MCP-1 mRNA expression profiles, determined by RT-PCR at 16 h, 1, 2, 3, and 7 days postphotocoagulation, were modified with aging. In 1-year-old mice (group IV), GFAP mRNA expression was already significantly higher than in the younger (10-12 week) group III before photocoagulation. After laser burns, GFAP mRNA expression peaked at 16-24 h and on day 7, decreasing thereafter. VEGF mRNA expression was markedly increased after photocoagulation in old mice eyes, reaching 2.7 times its basal level at day 3, while it was only slightly increased in young mice (1.3 times its level in untreated young mice 3 days postphotocoagulation). At all time points after photocoagulation, MCP-1 mRNA expression was elevated in old mice, reaching high levels of expression at 16 h and day 3 respectively. CONCLUSIONS: Our results were based on the study of four different age groups and included not only data from morphological observations but also from a molecular analysis of the various alterations of cytokine signaling and expression. One-year-old mice demonstrated more extensive CNV formation and a slower pace of regression after laser photocoagulation than younger mice. These were accompanied by differences in growth factors and cytokine expression profiles indicate that aging is a factor that aggravates CNV. The above results may provide some insight into possible therapeutic strategies in the future.

A larger spectrum of severe HIV-1--related disease in intravenous drug users in New York City.

Increasing mortality in intravenous (IV) drug users not reported to surveillance as acquired immunodeficiency syndrome (AIDS) has occurred in New York City coincident with the AIDS epidemic. From 1981 to 1986, narcotics-related deaths increased on average 32% per year from 492 in 1981 to 1996 in 1986. This increase included deaths from AIDS increasing from 0 to 905 and deaths from other causes, many of which were infectious diseases, increasing from 492 to 1091. Investigations of these deaths suggest a causal association with human immunodeficiency virus (HIV) infection. These deaths may represent a spectrum of HIV-related disease that has not been identified through AIDS surveillance and has resulted in a large underestimation of the impact of AIDS on IV drug users and blacks and Hispanics.

PlGF-1 and VEGFR-1 pathway regulation of the external epithelial hemato-ocular barrier. A model for retinal edema.

VEGF is considered as an important factor in the pathogenesis of macular edema. VEGF induces the rupture of the blood retinal barrier and may also influence the retinal pigment epithelial (RPE) outer retinal barrier. The aim of this work was to analyze the influence of the VEGF receptor pathways in the modulation of the RPE barrier breakdown in vitro and in vivo. The ARPE19 human junctions in culture are modulated by VEGF through VEGFR-1 but not through VEGFR-2. PlGF-1, that is a pure agonist of VEGFR-1, is produced in ARPE-19 cells under hypoxic conditions and mimics VEGF effects on the external retinal barrier as measured by TER and inulin flux. In vivo, the intravitreous injection of PlGF-1 induces a rupture of the external retinal barrier together with a retinal edema. This effect is reversible within 4 days. VEGF-E, that is a pure agonist of VEGFR-2, does not induce any acute effect on the RPE barrier. These results demonstrate that PlGF-1 can reproduce alterations of the RPE barrier occurring during diabetic retinopathy.

Plasmid electrotransfer of eye ciliary muscle: principles and therapeutic efficacy using hTNF-alpha soluble receptor in uveitis.

Due to its small size and particular isolating barriers, the eye is an ideal target for local therapy. Recombinant protein ocular delivery requires invasive and painful repeated injections. Alternatively, a transfected tissue might be used as a local producer of transgene-encoded therapeutic protein. We have developed a nondamaging electrically mediated plasmid delivery technique (electrotransfer) targeted to the ciliary muscle, which is used as a reservoir tissue for the long-lasting expression and secretion of therapeutic proteins. High and long-lasting reporter gene expression was observed, which was restricted to the ciliary muscle. Chimeric TNF-alpha soluble receptor (hTNFR-Is) electrotransfer led to elevated protein secretion in aqueous humor and to drastic inhibition of clinical and histological inflammation scores in rats with endotoxin-induced uveitis. No hTNFR-Is was detected in the serum, demonstrating the local delivery of proteins using this method. Plasmid electrotransfer to the ciliary muscle, as performed in this study, did not induce any ocular pathology or structural damage. Local and sustained therapeutic protein production through ciliary muscle electrotransfer is a promising alternative to repeated intraocular protein administration for a large number of inflammatory, degenerative, or angiogenic diseases.

Non-viral ocular gene therapy: potential ocular therapeutic avenues.

Non-viral vectors for potential gene replacement and therapy have been developed in order to overcome the drawbacks of viral vectors. The diversity of non-viral vectors allows for a wide range of various products, flexibility of application, ease of use, low-cost of production and enhanced "genomic" safety. Using non-viral strategies, oligonucleotides (ODNs) can be delivered naked (less efficient) or entrapped in cationic lipids, polymers or peptides forming slow release delivery systems, which can be adapted according to the organ targeted and the therapy purposes. Tissue and cell internalization can be further enhanced by changing by physical or chemical means. Moreover, a specific vector can be selected according to disease course and intensity of manifestations fulfilling specific requirements such as the duration of drug release and its level along with cells and tissues specific targeting. From accumulating knowledge and experience, it appears that combination of several non-viral techniques may increase the efficacy and ensure the safety of these evolving and interesting gene therapy strategies.

Intraocular implants for extended drug delivery: therapeutic applications.

An overview of ocular implants with therapeutic application potentials is provided. Various types of implants can be used as slow release devices delivering locally the needed drug for an extended period of time. Thus, multiple periocular or intraocular injections of the drug can be circumvented and secondary complications minimized. The various compositions of polymers fulfilling specific delivery goals are described. Several of these implants are undergoing clinical trials while a few are already commercialized. Despite the paramount progress in design, safety and efficacy, the place of these implants in our clinical therapeutic arsenal remains limited. Miniaturization of the implants allowing for their direct injection without the need for a complicated surgery is a necessary development avenue. Particulate systems which can be engineered to target specifically certain cells or tissues are another promising alternative. For ocular diseases affecting the choroid and outer retina, transscleral or intrasscleral implants are gaining momentum.

Evaluation of a topical cyclosporine A prodrug on corneal graft rejection in rats.

PURPOSE: To assess the efficacy of a topical cyclosporine A (CsA), water-soluble prodrug, for promoting the survival of allogenic rat corneal grafts after penetrating keratoplasty (PKP). METHODS: Corneas of Brown-Norway rats (donors) were transplanted to Lewis rats (recipients). Transplanted rats were divided in three treatment groups: group I (PBS) and group II (0.26% Debio088) received drops five times per day. Group III received a daily intramuscular CsA injection (10 mg/kg/day). Blood CsA concentrations were measured on days 2 and 14. On day 4, 10, 13 after PKP, grafts were scored for corneal transparency, edema and extent of neovascularization. An opacity score of greater than or equal to 3 was considered as a nonreversible graft rejection process. On day 14, the experimental eyes were processed for histology. RESULTS: On day 13, 12 of the 18 corneal transplants (67%) in group I showed irreversible graft rejection. Three of 18 transplants (19%) in group II and 5 of 16 transplants (28%) in group III showed irreversible graft rejection (p=0.013/p=0.019, OR=0.14/0.06 versus vehicle). Each mean clinical score for edema, opacity, and neovessels in group II were significantly lower than those of the grafts in group I (respectively p=0.010, p=0.013, p=0.024) and III except for neovessels (respectively p=0.002, p=0.001, p=0.057). Histology confirmed the clinical results. The mean CsA blood levels for groups II and III were, respectively 54+/-141 mug/l and 755+/-319 mug/l on day 2 and 14+/-34 mug/l and 1318+/-463 mug/l on day 14. CONCLUSIONS: Debio088 CsA prodrug drops given five times daily are as effective as intramuscular injection of 10 mg/kg/day for the prevention of acute corneal graft rejection in rats.

Uveitis in children and adolescents.

AIMS: To study the relative occurrence of uveitis (intraocular inflammation) and its causes in children and adolescents. METHODS: Patients with uveitis examined and followed during a period of 10 years were categorised by age and sex. All underwent ocular examination and an individually tailored battery of laboratory tests. The intraocular manifestations were classified according to the anatomical location of the inflammation and their most probable cause. The final diagnosis was based on typical clinical ocular and extraocular symptoms and signs and on the results of specific laboratory investigations. RESULTS: Out of 821 patients, 276 (33.1%) were 18 years of age or younger with a male to female ratio of 1 to 1. In these 276 children and adolescents, 70.3% had bilateral ocular involvement. Intermediate uveitis was the most frequent anatomical diagnosis. In many cases, symptoms were mild despite the prominent signs and marked decrease of vision. The underlying cause for the uveitis was evaluated as non-infectious in 184 cases (66.7%) and infectious in 92 cases (33.3%). A potential aetiology and/or a definite clinical diagnosis were established in 74.6% of the cases and only 25.4% of the 276 patients were classified as idiopathic. Juvenile idiopathic arthritis (JIA) was the most common systemic disease association diagnosed in 14.9% of these children. Parasite infestation was the most common infectious association. CONCLUSIONS: Uveitis in children and adolescents is not as low as previously reported. Parasite infestation on the one hand and JIA on the other hand are the most common aetiologies associated with the uveitis in these young patients.

Prospective study of infections in indwelling central venous catheters using quantitative blood cultures.

PURPOSE: Surgically implanted central venous catheters are widely used in cancer patients in whom there is a need for prolonged venous access for chemotherapy, parenteral nutrition, antibiotics, and blood sampling. This study evaluated catheter infectious complications, including catheter-related sepsis, exit site infection, and tunnel infection. Specifically, an evaluation of the incidence, type, and response to treatment of indwelling catheter infections was performed, and conditions under which the catheter should be removed were delineated. PATIENTS AND METHODS: During the year of this study, 488 central venous catheters were implanted. Records were maintained on demographic variables, date of catheter implantation, surgeon, white blood cell count, absolute neutrophil count, and underlying diagnosis. Blood for both aerobic and anaerobic culture was collected from each patient. For patients in whom infection developed, clinical features, white blood cell count, absolute neutrophil count, and microbiologic data were noted, as were the clinical course and response to treatment. RESULTS: A total of 142 episodes of infectious complications were documented. There were 88 episodes of catheter-related sepsis, and 33 of 54 evaluable episodes (61 percent) were successfully treated with antibiotics. There were 34 episodes of exit site infection, and 20 of the 29 evaluable episodes (69 percent) were successfully treated with antibiotics and local care. Of the 20 tunnel infections, only five (25 percent) were successfully treated with antibiotics, and the other 15 required catheter removal for cure. Twelve of the 15 cases requiring catheter removal were caused by Pseudomonas species. CONCLUSION: On the basis of these results, compulsory removal of the catheter is not required in cases of catheter-related sepsis. Similarly, exit site infections can often be cured by means of antibiotics and local care. However, catheter removal is required to achieve cure in most tunnel infections, particularly if Pseudomonas species are cultured from the exit sites of patients with tunnel infection.

A microculture technique for the evaluation of corneal cell metabolism in vitro.

A microculture technique for the evaluation of the metabolic activity of corneal cells is described and analyzed. The extent of DNA synthesis in microcultures with 10(3) to 2.5 X 10(3) cells per well was initially low during day 1, increasing steadily thereafter. Higher initial concentration of 10(4) to 2 X 10(4) cells per microculture demonstrated a high metabolic activity during days 1 and 2 in culture, followed by a rapid and marked decrease on days 3 and 4. The origin and concentration of serum in the system have been found to be crucial. Xenogeneic serum (fetal calf serum--FCS) had the most potent stimulatory effect on DNA and protein synthesis. Syngeneic serum (guinea pig serum, strain 13--SGpS) or allogeneic serum (guinea pig serum strain 2--AGpS) had a generally less stimulatory effect on the metabolic activity. However, both sera had a relatively much stronger effect on the protein synthesis.

Stimulation of keratocyte metabolism by products of lymphoid cells.

Products of leukocytes were found to activate cultures of keratocytes, manifested by the increased incorporation of thymidine or leucine. The keratocyte activation capacity crosses the species barriers between rabbit, monkey, and human. The level of secreted keratocyte-activating factor(s) (KAF) depends on the stimulation of the leukocytes. Thus unstimulated rabbit leukocytes produced very little or no KAF, whereas significant levels were produced by leukocytes stimulated with lipopolysaccharide (LPS) or concanavalin A. High levels of KAF were also found in supernatants of human mononuclear leukocytes stimulated by LPS. The effects of the activated leukocyte supernatants on keratocyte metabolism resembled the increased metabolic activity induced by the fibroblast and epidermal growth factors. The relationship between KAF and a possible modulating role of the products of lymphoid cells on corneal wound healing is suggested.

Ocular penetration of cyclosporin A. The rabbit eye.

In the rabbit, after oral ingestion of 20 mg/kg/day of cyclosporin A (CsA) a high level of the drug is found in the blood. This level increases steadily during the first 3 days, leveling off by days 5-7. Although the blood level of CsA was within the "therapeutic window" of 400-600 ng/ml, the drug was not detected within the ocular tissues. Local application using 2% CsA in olive oil induced a high concentration of the drug in the cornea and conjunctive but no detectable levels within the intraocular structures. When a significant intraocular inflammation is induced in one of the rabbit eyes, CsA ingested orally reaches detectable levels in nearly all tissues of the inflamed eyes. Highest concentrations of the drug were observed within the chorioretinal complex in these eyes. In the contralateral (noninflamed) eyes, however, no detectable CsA was found either intraocularly or extraocularly. In the significantly inflamed eyes, local application of the drug induced a high CsA level within the anterior segment only, without any detectable levels within the choroid and retina.

Dissociation between humoral and cellular immune responses to lens antigens.

Rabbits immunized against lens extracts from various other (xenogeneic) species reacted well with the immunizing lens extracts by immunological assays measuring either humoral or cellular immune responses. A dissociation was observed, however, between the humoral and cellular immune responses when lenses from rabbit (allogeneic) or from nonimmunizing species were tested. Antibodies from these rabbits cross-reacted with lenses from rabbit or all other tested species, with levels similar to those obtained with the immunizing lens extracts. The antibodies were determined by both serological tests and the Arthus skin reaction. On the other hand, these rabbits failed to cross-react with rabbit or nonimmunizing animal lens extracts by the cell-mediated delayed skin reaction, and their lymphocytes reacted poorly in culture with the nonimmunizing lenses. A similar dissociation between the humoral and cellular responses was found in rabbits immunized against allogeneic lens. Most of these rabbits produced moderate levels of lens antibodies demonstrated by the serological tests and the Arthus skin reaction but failed to exhibit delayed-type hypersensitivity, and their lymphocyte cultures reacted poorly or not at all to the rabbit or other lenses. The results are discussed in view of their relevance to the hypotheses concerning the pathogenesis of phacogenic uveitis.

Thrombospondin and in vivo angiogenesis induced by basic fibroblast growth factor or lipopolysaccharide.

PURPOSE: To reexamine the possible effect of human thrombospondin on in vivo angiogenesis. METHODS: In vivo angiogenesis in the rabbit cornea was induced by implants of Elvax-40 sequestering human recombinant basic fibroblast growth factor (bFGF) or bacterial endotoxin lipopolysaccharide (LPS). Implants sequestering various concentrations of thrombospondin were examined for their ability to induce angiogenesis and also for their possible influence on the angiogenic potential of bFGF- or LPS-sequestering implants. RESULTS: Constant and reproducible angiogenic stimuli were obtained with implants sequestering 250 ng or more of bFGF or 100 ng or more of LPS. Implants sequestering 500 ng of thrombospondin induce very little clinical change but larger concentrations induce infiltration of leukocytes and a mild angiogenic stimulus. Combination of thrombospondin implants with bFGF or LPS implants enhanced the angiogenic response to either of these factors. The thrombospondin enhancing effect was more prominent when LPS was the stimulating factor. Histologic examination of the tested corneas disclosed that the LPS angiogenic stimulus follows the influx of leukocytes. Conversely, the bFGF angiogenic stimulus appears to be associated with the proliferation of stromal keratocytes and corneal epithelial cells. The thrombospondin angiogenic enhancing effect on both the LPS and bFGF stimuli was correlated with an increased infiltration of polymorphonuclear cells. CONCLUSION: In this system, thrombospondin enhanced the in vivo angiogenic process induced by bFGF or LPS. This enhancement appears to be associated with an in vivo activation and chemotactic effect on the polymorphonuclear cells.

Topical formulations of novel angiostatic steroids inhibit rabbit corneal neovascularization.

PURPOSE: To evaluate the antiangiogenic potential of topical ophthalmic formulations of the novel angiostatic steroids AL-3789 and AL-4940, using a rabbit model of corneal neovascularization. METHODS: Neovascularization was induced in the rabbit cornea by surgical implantation of a standard ethylene vinyl acetate copolymer (Elvax-40) pellet containing 1 microg lipopolysaccharide. Coded formulations of the control vehicle or the following test agents were administered in prevention and intervention treatment protocols: 1% formulations of AL-3789, AL-4940, and cortisol acetate as a positive drug control. Three doses of AL-3789 (0.01%, 0.1%, and 1%) were also evaluated in a prevention treatment protocol. Corneal responses were monitored throughout a 2-week treatment period, and 1 week after the last treatment dose. Observations included quantitative measurement of the area of new blood vessel growth and qualitative assessment of cellular infiltrate and edema. All treatments and observations were performed in a double-masked manner. RESULTS: All tested formulations, except the vehicle and the 0.01% AL-3789 preparation, significantly inhibited corneal neovascularization and other lipopolysaccharide-induced responses in the various treatment protocols employed. AL-4940, the free alcohol form of AL-3789, was slightly less effective than cortisol acetate or AL-3789. The extent of inhibition of the angiogenic response by the 1% and 0.1% AL-3789 suspensions ranged from 76% to 100% 1 week after the last treatment. CONCLUSIONS: The antiangiogenic steroid AL-3789 may be a therapeutically useful angiostatic agent for corneal neovascularization and potentially could be effective in other ocular neovascular diseases.

Monoclonal antibodies to Müller's cells of the retina.

The immunohistologic properties of two monoclonal antibodies produced by hybridomas generated from bovine retinal S-antigen (S-Ag) immunized mice were investigated. These monoclonal antibodies demonstrated a low antibody titer to the original S-Ag preparation by the ELISA method. Immunohistologic studies using avidin-biotin-peroxidase complex (ABC) showed strong specific binding to the retinal Müller cells of all species tested (human, bovine, guinea pig and rat), a weaker binding to cell bodies and proximal component of the outer segments of the photoreceptor, but no apparent binding to the distal component of the outer segments of the photoreceptor cells.

Syndrome of brittle cornea, blue sclera, and joint hyperextensibility.

In 3 families 4 patients were affected with the syndrome of brittle cornea, blue sclera, and hyperextensible joints (brittle cornea syndrome). From the review of 17 affected patients described previously and our cases, it appears that this rare autosomal recessive syndrome has variable expressivity. Two different groups of patients may be distinguished: one includes 5 families, all of Tunisian Jewish origin. All patients in this group also have red hair. In the second group, 9 families are from various ethnic origins; affected patients in this group have a normal distribution of hair color. A possible explanation for the existence of these 2 different group of patients is that the locus of the gene responsible for the syndrome is closely linked to the locus for a gene responsible for hair color with linkage disequilibrium in Tunisian Jews (Sepharadim).

Autosomal recessive colobomatous microphthalmia.

Colobomatous microphthalmia was studied in multiple relatives of 5 families. In these families, the disorder was an autosomal recessive trait as opposed to the usual autosomal dominant form of the disorder. A relatively high incidence of this recessive allele is found in the Iranian Jewish community.

Experimental specific memory reaction to cornea, lens, and retina antigens.

Lymphocytes from rabbits immunized against various ocular extracts were challenged in vitro by the immunizing extract, by other ocular extracts from the same species, and by analogous extracts from other species. Immunological memory reaction, demonstrated by the blast transformation phenomenon, was found toward organ-specific and species-specific antigens. The organ cross-reactivity was greatest when cornea and lens were tested. Immunization of rabbits against calf cornea with extract emulsified in Freund adjuvant injected into the hind foot-pads induced the appearance of specific memory cells in popliteal lymph nodes, spleen, and blood. Immunization by interlamellar xenografting has shown that memory lymphocytes are mainly localized in the preauricular lymph nodes and are nearly absent from spleen and popliteal lymph nodes. These findings indicate the important role of the local immunological mechanism of the eye.