XIAP Protection of Photoreceptors in Animal Models of Retinitis Pigmentosa

BACKGROUND: Retinitis pigmentosa (RP) is a blinding genetic disorder that is caused by the death of photoreceptors in the outer nuclear layer of the retina. To date, 39 different genetic loci have been associated with the disease, and 28 mutated genes have been identified. Despite the complexity of the underlying genetic basis for RP, the final common pathway is photoreceptor cell death via apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: In this study, P23H and S334ter rhodopsin transgenic rat models of RP were used to test the neuroprotective effects of anti-apoptotic gene therapy. Adeno-associated viruses (AAV) carrying the X-linked inhibitor of apoptosis (XIAP) or green fluorescent protein (GFP) were delivered subretinally into the eye of transgenic rat pups. Histological and functional measures were used to assess neuroprotection. XIAP is known to block apoptosis by inhibiting the action of caspases-3, -7 and -9. The results show that XIAP gene therapy provides long-term neuroprotection of photoreceptors at both structural and functional levels. CONCLUSIONS/SIGNIFICANCE: Our gene therapy strategy targets the apoptotic cascade, which is the final common pathway in all forms of retinitis pigmentosa. This strategy holds great promise for the treatment of RP, as it allows for the broad protection of photoreceptors, regardless of the initial disease causing mutation

Using the X-linked inhibitor of apoptosis (XIAP) as a therapeutic agent in rodent models of retinal degeneration

Retinitis Pigmentosa (RP) is the leading cause of hereditary blindness. The clinical manifestations of RP are night blindness, loss of peripheral vision leading to tunnel vision and a decreased electroretinogram (ERG) amplitude. There are numerous genes that when mutated cause RP. Regardless of the causative mutation, the end point is the same, photoreceptor cell death by apoptosis or programmed cell death (PCD). Interestingly, though all photoreceptors in an affected individual would have a mutation, disease progresses at a relatively slow rate indicating the affected cells can function. It is unclear as to why completely unrelated mutations would cause photoreceptors to die via the same mechanism. Somatic gene replacement studies using viral vectors in animal models have been successful in slowing down disease. However, attempting to cure all retinal degeneration mutations by this approach would be costly and difficult for two reasons. First, there are some mutations that comprise a small percentage of all RP cases and the cost/benefit ratio to develop a therapeutic agent for a few people would be enormous. Secondly, a number of genes that cause retinal degenerations have not been cloned. A more practical approach involves preventing apoptosis of the photoreceptor cell. The advantage of this approach is that the causative mutation does not need to be known. Developing a single therapeutic agent to target RP would also be economically more favourable than developing multiple therapeutic agents. The X-linked Inhibitor of Apoptosis (XIAP) can inhibit cell death from numerous triggers both in vitro and in vivo. XIAP prevents apoptosis by binding to and inhibiting caspase-3, -7 and -9. Work presented here shows that adeno-associated virus (AAV) encoding XIAP is neuroprotective when injected sub-retinally in an acute retinal degeneration model in Sprague Dawley rats. In addition, transgenic mice overexpressing XIAP throughout the neural retina have normal retinal morphology and their ERGs appear normal. These findings suggest that XIAP holds promise as a therapeutic agent in the treatment of hereditary retinal diseases such as retinitis pigmentosa

New concepts in the diagnosis and pathogenesis of Trichomonas vaginalis

Trichomonas vaginalis infection is the most commonly encountered sexually transmitted disease. There is a need for more accurate and rapid laboratory diagnostic methods, leading to better control and treatment strategies. Various virulence factors such as adherence, contact-independent factors, hemolysis and acquisition of host macromolecules have been shown to play a role in the pathogenesis of this infection. Detection of the factors that are only present in the pathogenic isolates of trichomonads will lead to a better understanding of the epidemiology of this pathogen. Culture technique is highly specific compared with microscopic techniques, but it is time consuming. Immunological techniques lack proper correlation with clinical manifestations. The application of monoclonal antibodies, either singly or in a group that recognizes a common antigen, along with methods such as detection of common DNA fragment from clinical specimens, may have a promising future in the laboratory diagnosis of trichomoniasis

Structural and functional protection of photoreceptors from MNU-induced retinal degeneration by the X-linked inhibitor of apoptosis

Purpose: To evaluate the neuroprotective effects of adenoassociated virus delivery of XIAP in N-methyl-N-nitrosourea (MNU)-induced retinal degeneration in Sprague-Dawley rats.Methods: Sprague-Dawley rats were injected subretinally with recombinant adenoassociated virus (rAAV) encoding either XIAP or green fluorescent protein (GFP; injection control). Six weeks after injection, the animals received an intraperitoneal injection of MNU, a DNA methylating agent, at a dose of 60 mg/kg. Electroretinograms (ERGs) were recorded at 0, 24, 48 and 72 hours and 1 week after MNU. The rats were killed after the ERG was performed and were perfused with 4% paraformaldehyde. Eyes were then enucleated and embedded for cryosectioning. Eye sections were analyzed by TUNEL and histologic techniques. Real-time PCR and Western analysis were performed to confirm the overexpression of XIAP in injected eyes.Results: Real-time PCR and Western analysis confirmed the overexpression of XIAP in virus-injected eyes in comparison to uninjected control eyes. At 24 hours after MNU injection, fewer cells had undergone apoptosis in the XIAP-treated eyes in comparison with GFP-injected or uninjected eyes. Hematoxylin and eosin staining revealed that the uninjected and GFP-injected photoreceptors were destroyed by 72 hours after injection of MNU, whereas the AAV-XIAP-injected eyes showed structural protection of the photoreceptors at all time points throughout the 1-week sampling period. ERGs showed functional protection up to 1 week after MNU injection in the AAV-XIAP-injected eye, whereas no response was observed in the control eye.Conclusions: The results suggest that XIAP is protective against this potent chemotoxic agent and holds promise as a therapeutic agent in gene therapy approaches to treating retinitis pigmentosa

Abnormalities of the vitreoretinal interface caused by dysregulated Hedgehog signaling during retinal development

Mutations in Patched (PTCH), encoding the Hedgehog (Hh) receptor, underlie Basal Cell Naevus syndrome (BCNS) and, in addition to tumor predisposition, are associated with a wide range of \u27patterning\u27 defects. The basis for the underlying patterning problems in Hh-dependent tissues in BCNS and their long-term consequences on tissue homeostasis are, however, not known. Hh signaling is required for normal growth and organization of the mammalian retina and we show that PtchlacZ+/- mice exhibit vitreoretinal abnormalities resembling those found in BCNS patients. The retinas of PtchlacZ+/- mice exhibit abnormal cell cycle regulation, which culminates in photoreceptor dysplasia and Müller cell-derived gliosis. In BCNS, the intraretinal glial response results in epiretinal membrane (ERM) formation, a proliferative and contractile response on the retinal surface. ERMs are a cause of significant visual loss in the general, especially elderly, population. We hypothesize that alteration of Müller cell Hh signaling may play a role in the pathogenesis of such age-related \u27idiopathic\u27 ERMs