Glucocorticoids induce retinal toxicity through mechanisms mainly associated with paraptosis.

PURPOSE: Corticosteroids have recorded beneficial clinical effects and are widely used in medicine. In ophthalmology, besides their treatment benefits, side effects, including ocular toxicity have been observed especially when intraocular delivery is used. The mechanism of these toxic events remains, however, poorly understood. In our present study, we investigated the mechanisms and potential pathways of corticosteroid-induced retinal cell death. METHODS: Rats were sacrificed 24 h and 8 days after an intravitreous injection of 1 microl (40 microg) of Kenacort Retard. The eyes were processed for ultra structure analysis and detection of activated caspase-3, cytochrome-C, apoptosis-inducing factor (AIF), LEI-L-Dnase II, terminal transferase dUTP nick end labeling (TUNEL), and microtubule-associated protein 1-light chain 3 (MAP-LC3). In vitro, rat retinal pigment epithelial cells (RPE), retinal Müller glial cells (RMG) and human ARPE-19 cells were treated with triamcinolone acetonide (TA) or other glucocorticoids. Cell viability was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5 phenyltetrazolium bromide test (MTT) assay and cell counts. Nuclei staining, TUNEL assay, annexin-V binding, activated caspase-3 and lactate dehydrogenase (LDH) production characterized cell death. Localization of cytochrome-C, AIF, LEI-and L-Dnase II, and staining with MAP-LC3 or monodansylcadaverine were also carried out. Finally, ARPE-19 cells transfected with AIP-1/Alix were exposed to TA. RESULTS: In vitro incubation of retinal cell in the presence of corticosteroids induced a specific and dose-dependent reduction of cell viability. These toxic events were not associated with the anti-inflammatory activity of these compounds but depended on the hydro solubility of their formulation. Before cell death, extensive cytoplasmic vacuolization was observed in the retinal pigment epithelial (RPE) cells in vivo and in vitro. The cells however, did not show known caspase-dependent or caspase-independent apoptotic reactions. These intracellular vacuoles were negative for MAP-LC3 but some stained positive for monodansylcadaverine. Furthermore, over expression of AIP-1/Alix inhibited RPE cell death. CONCLUSIONS: These observations suggest that corticosteroid-induced retinal cell death may be carried out mainly through a paraptosis pathway.

Species and Strain-specific Typing of Cryptosporidium Parasites in Clinical and Environmental Samples

Cryptosporidiosis has recently attracted attention as an emerging waterborne and foodborne disease as well as an opportunistic infection in HIV infected individuals. The lack of genetic information, however, has resulted in confusion in the taxonomy of Cryptosporidium parasites and in the development of molecular tools for the identification and typing of oocysts in environmental samples. Phylogenetic analysis of the small subunit ribosomal RNA (SSU rRNA) gene has shown that the genus Cryptosporidium is comprised of several distinct species. Our data show the presence of at least four species: C. parvum, C. muris, C. baileyi and C. serpentis (C. meleagridis, C. nasorum and C. felis were not studied). Within each species, there is some sequence variation. Thus, various genotypes (genotype 1, genotype 2, guinea pig genotype, monkey genotype and koala genotype, etc.) of C. parvum differ from each other in six regions of the SSU rRNA gene. Information on polymorphism in Cryptosporidium parasites has been used in the development of species and strain-specific diagnostic tools. Use of these tools in the characterization of oocysts various samples indicates that C. parvum genotype 1 is the strain responsible for most human Cryptosporidium infections. In contrast, genotype 2 is probably the major source for environmental contamination of environment, and has been found in most oysters examined from Chesapeake Bay that serve as biologic monitors of surface water. Parasites of Cryptosporidium species other than C. parvum have not been detected in HIV+ individuals, indicating that the disease in humans is caused only by C. parvum.

Long-term stability of retinal function despite retained intraocular metallic foreign body.

BACKGROUND: Persisting metallic intraocular foreign bodies (IOFB) with a ferrous content have been associated with ocular siderosis and retinal degeneration. We describe two patients in whom a metallic IOFB containing iron was left embedded for many years in the choroid and sclera after having penetrated through the vitreous and the retina. HISTORY AND SIGNS: Two male patients, aged 41 and 48 years, presented with a metallic IOFB sustained during a work accident involving metal tools. THERAPY AND OUTCOME: For the first patient it was deemed unwise to operate, as the IOFB was also lodged very deeply in the choroid and sclera in the inferior temporal quadrant. The second patient underwent pars plana vitrectomy, but the IOFB could not be removed surgically as it was too deeply embedded in the sclera and choroid. After a period of 6 years (Case 1) and 4 years (Case 2) of follow-up, visual acuity remained at 1.0 and the IOFB was encased in a fibrotic capsule in both cases. Full-field and multifocal electroretinograms showed an inter-ocular asymmetry at baseline, which remained stable during the follow-up. CONCLUSIONS: Ocular siderosis may not develop in patients with a deeply embedded metallic IOFB. Regular monitoring of both visual function and the electroretinogram is mandatory when the IOFB is left inside the eye.

Molecular genetics of "PRPF31" dominant mutations linked to retinitis pigmentosa

ABSTRACT : The retina is one of the most important human sensory tissues since it detects and transmits all visual information from the outside world to the brain. Retinitis pigmentosa (RP) is the name given to a group of inherited diseases that affect specifically the photoreceptors present in the retina and in many instances lead to blindness. Dominant mutations in PRPF31, a gene that encodes for a pre-mRNA splicing factor, cause retinitis pigmentosa with reduced penetrance. We functionally investigated a novel mutation, identified in a large family with autosomal dominant RP, and 7 other mutations, substitutions and microdeletions, in 12 patients from 7 families with PRPF31-linked RP. Seven mutations lead to PRPF31 mRNA with premature stop codons and one to mRNA lacking the exon containing the initiation codon. Quantification of PRPF31 mRNA and protein levels revealed a significant reduction in cell lines derived from patients, compared to non carriers of mutations in PRPF31. Allelic quantification of PRPF31 mRNA indicated that the level of mutated mRNA is very low compared to wild-type mRNA. No mutant protein was detected and the subnuclear localization of wild-type PRPF31 remains the same in cell lines from patients and controls. Blocking nonsense-mediated mRNA decay in cell lines derived from patients partially restored PRPF31 mutated mRNA but derived proteins were still undetectable, even when protein degradation pathways were inhibited. Our results demonstrated that the vast majority of PRPF31 mutations result in null alleles, since they are subject to surveillance mechanisms that degrade mutated mRNA and possibly block its translation. Altogether, these data indicate that the likely cause of PRPF31-linked RP is haploinsufficiency, rather than a dominant negative effect. Penetrance of PRPF31 mutations has been previously demonstrated to be inversely correlated with the level of PRPF31 mRNA, since high expression of wild-type PRPF31 mRNA protects from the disease. Consequently, we have investigated the genetic modifiers that control the expression of PRPF31 by quantifying PRPF31 mRNA levels in cell lines derived from 200 individuals from 15 families representative of the general population. By linkage analyses we identified a 8.2Mb-region on chromosome 14q21-23 that contains a gene involved in the modulation of PRPF31 expression. We also assessed apreviously-mapped penetrance factor invariably located on the wild-type allele and linked to the PRPF31 locus in asymptomatic patients from different families with RP. We demonstrated that this modifier increases the expression of both PRPF31 alleles already at the pre-mRNA level. Finally, our data suggest that PRPF31 mRNA expression and consequently the penetrance of PRPF31 mutations is modulated by at least 2 diffusible compounds, which act on both PRPF31 alleles during their transcription.

Ocular distribution, spectrum of activity, and in vivo viral neutralization of a fully humanized anti-herpes simplex virus IgG Fab fragment following topical application.

Herpes simplex ocular infection is a major cause of corneal blindness. Local antiviral treatments exist but are associated with corneal toxicity, and resistance has become an issue. We evaluated the biodistribution and efficacy of a humanized anti-herpes simplex virus (anti-HSV) IgG FAb fragment (AC-8; 53 kDa) following repeated topical administration. AC-8 was found in the corneal epithelium, anterior stroma, subepithelial stromal cells, and retinal glial cells, with preferential entry through the ocular limbus. AC-8 was active against 13 different strains of HSV-1, with 50% and 90% mean effective concentrations (MEC(50) and MEC(90), respectively) ranging from 0.03 to 0.13 μg/ml, indicating broad-spectrum activity. The in vivo efficacy of AC-8 was evaluated in a mouse model of herpes-induced ocular disease. Treatment with low-dose AC-8 (1 mg/ml) slightly reduced the ocular disease scores. A greater reduction of the disease scores was observed in the 10-mg/ml AC-8-treated group, but not as much as with trifluridine (TFT). AC-8 treatment reduced viral titers but less than trifluridine. AC-8 did not display any toxicity to the cornea or other structures in the eye. In summary, topical instillation of an anti-HSV FAb can be used on both intact and ulcerated corneas. It is well tolerated and does not alter reepithelialization. Further studies to improve the antiviral effect are needed for AC-8 to be considered for therapeutic use.

Acquired and Congenital Ocular Toxoplasmosis Experimentally Induced in Calomys callosus (Rodentia, Cricetidae)

An experimental model for acquired and congenital ocular toxoplasmosis as well as a model to induce experimental autoimmune uveitis (EAU) was investigated in Calomys callosus. Toxoplasma gondii, ME-49 strain, was used to infect males and pregnant- and not pregnant-females while S-antigen, a major glycoprotein of the retinal photoreceptor cell, was used to induce EAU. The ocular lesions elicited by T. gondii were characterized by the presence of cysts, free tachyzoites and inflammatory cells in the retina or related tissues. In the congenital form, 40% of the fetus presented ocular lesions, i.e., presence of cysts in the retina, vitreous, and extra-retinal tissues. In the acquired form, 75% of the females and 50% of the males presented unilateral ocular cysts both at 21 and 47 days post-infection. It was also demonstrated that S-antigen was not uveitogenic in the C. callosus model. No lesion was observed in the animals exclusively immunized with this retinal component, even when jacalin was used as additional adjuvant for polyclonal response to the retinal antigen. It can be concluded that C. callosus may constitute in a promising model for study both acquired and congenital ocular toxoplasmosis, particularly when it is important to make sure that a non autoimmune process is involved in the genesis of the ocular infection.

MAP kinase pathways in UV-induced apoptosis of retinal pigment epithelium ARPE19 cells.

The retinal pigment epithelium (RPE) is constantly exposed to external injuries which lead to degeneration, dysfunction or loss of RPE cells. The balance between RPE cells death and proliferation may be responsible for several diseases of the underlying retina, including age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR). Signaling pathways able to control cells proliferation or death usually involve the MAPK (mitogen-activated protein kinases) pathways, which modulate the activity of transcription factors by phosphorylation. UV exposure induces DNA breakdown and causes cellular damage through the production of reactive oxygen species (ROS) leading to programmed cell death. In this study, human retinal pigment epithelial cells ARPE19 were exposed to 100 J/m(2) of UV-C and MAPK pathways were studied. We first showed the expression of the three major MAPK pathways. Then we showed that activator protein-1 (AP-1) was activated through phosphorylation of cJun and cFos, induced by JNK and p38, respectively. Specific inhibitors of both kinases decreased their respective activities and phosphorylation of their nuclear targets (cJun and cFos) and reduced UV-induced cell death. The use of specific kinases inhibitors may provide excellent tools to prevent RPE apoptosis specifically in RPE diseases involving ROS and other stress-related compounds such as in AMD.

OCT e índices de insulinorresistencia en diabéticos tipo 2 no insulinodependientes sin signos de retinopatía

A una població de diabètics tipus 2 no insulinodepenents i sense signes clínics de retinopatia diabètica, realitzem un estudi de la possible relació entre índexs indirectes d’insulinorresistència i troballes de tomografia de coherència òptica de gruix macular y de gruix de la capa de fibres nervioses de la retina peripapil•lar. La nostra hipòtesi consisteix en valorar el possible dany neurodegeneratiu previ a les alteracions microvasculars clàssiques sobre la retina a aquesta malaltia i estudiar la seva possible relació amb la insulinorresistència.

Utilització de Bevacizumab en patologia diferent de la degeneració macular associada a l’edat

INTRODUCCIÓ L’angiogènesi és el procés que condueix a la formació de nous vasos sanguinis a partir de la vascularització preexistent. Disposem en l’actualitat de diversos fàrmacs que inhibeixen un factor proangiogènic, el factor de creixement de l’endoteli vascular (VEGF). L’objectiu del nostre estudi és determinar l’efectivitat de la mol·lècula bevacizumab (Avastin ®) en el tractament de diverses patologies amb neovascularització i/o edema macular. MATERIAL I MÈTODES Estudi prospectiu obert no randomitzat. Àmbit d’estudi: pacients del Servei d’Oftalmologia de l’Hospital Municipal de Badalona. Població a estudi: pacients amb Oclusió venosa de l’artèria central de la retina i oclusió de branca venosa / Edema macular diabètic / Edema macular uveític / Retinopatia diabètica proliferativa / Glaucoma neovascular / Metàstasi a iris de tumor oat cell de pulmó Metodologia: comparació de l’espessor macular central o de la neovascularització, abans i després del tractament RESULTATS En tots els subgrups a estudi s’observa millora del paràmetre estudiat globalment. Pacients amb edema macular diabètic: milloren agudesa visual 0’22 de mitjana, 169 micres de disminució promig de l’edema. Pacients amb edema macular secundari a oclusió venosa: milloren agudesa visual 0’23 de mitjana, 143 micres de disminució promig de l’edema. Pacients amb edema macular uveític: milloren agudesa visual 0’22 de mitjana, amb una disminució promig de l’edema macular de 263 micres. Pacients amb retinopatia diabètica, glaucoma neovascular o metàstasi iridiana: regressió parcial/completa de la neovascularització. CONCLUSIONS El nostre estudi suggereix que el bevacizumab intravitri és una teràpia eficaç i segura en casos d’edema macular o neovascularització de diversa etiologia diferent de DMAE, encara que d’efecte temporal. Cal tenir en compte els riscos locals i sistèmics, així com el seu ús compassiu. Serien necessaris estudis prospectius aleatoris més extensos i amb major temps de seguiment per reforçar els resultats favorables actuals d’aquest tractament.

Chromatic pupil responses: preferential activation of the melanopsin-mediated versus outer photoreceptor-mediated pupil light reflex.

OBJECTIVE: To weight the rod-, cone-, and melanopsin-mediated activation of the retinal ganglion cells, which drive the pupil light reflex by varying the light stimulus wavelength, intensity, and duration. DESIGN: Experimental study. PARTICIPANTS: Forty-three subjects with normal eyes and 3 patients with neuroretinal visual loss. METHODS: A novel stimulus paradigm was developed using either a long wavelength (red) or short wavelength (blue) light given as a continuous Ganzfeld stimulus with stepwise increases over a 2 log-unit range. The pupillary movement before, during, and after the light stimulus was recorded in real time with an infrared illuminated video camera. MAIN OUTCOME MEASURES: The percent pupil contraction of the transient and sustained pupil response to a low- (1 cd/m(2)), medium- (10 cd/m(2)), and high-intensity (100 cd/m(2)) red- and blue-light stimulus was calculated for 1 eye of each subject. From the 43 normal eyes, median and 25th, 75th, 5th, and 95th percentile values were obtained for each stimulus condition. RESULTS: In normal eyes at lower intensities, blue light evoked much greater pupil responses compared with red light when matched for photopic luminance. The transient pupil contraction was generally greater than the sustained contraction, and this disparity was greatest at the lowest light intensity and least apparent with bright (100 cd/m(2)) blue light. A patient with primarily rod dysfunction (nonrecordable scotopic electroretinogram) showed significantly reduced pupil responses to blue light at lower intensities. A patient with achromatopsia and an almost normal visual field showed selective reduction of the pupil response to red-light stimulation. A patient with ganglion cell dysfunction owing to anterior ischemic optic neuropathy demonstrated global loss of pupil responses to red and blue light in the affected eye. CONCLUSIONS: Pupil responses that differ as a function of light intensity and wavelength support the hypothesis that selected stimulus conditions can produce pupil responses that reflect phototransduction primarily mediated by rods, cones, or melanopsin. Use of chromatic pupil responses may be a novel way to diagnose and monitor diseases affecting either the outer or inner retina.

PKCζ Mediates Breakdown of Outer Blood-Retinal Barriers in Diabetic Retinopathy.

AIMS/HYPOTHESIS: Diabetic macular edema represents the main cause of visual loss in diabetic retinopathy. Besides inner blood retinal barrier breakdown, the role of the outer blood retinal barrier breakdown has been poorly analyzed. We characterized the structural and molecular alterations of the outer blood retinal barrier during the time course of diabetes, focusing on PKCζ, a critical protein for tight junction assembly, known to be overactivated by hyperglycemia. METHODS: Studies were conducted on a type2 diabetes Goto-Kakizaki rat model. PKCζ level and subcellular localization were assessed by immunoblotting and immunohistochemistry. Cell death was detected by TUNEL assays. PKCζ level on specific layers was assessed by laser microdissection followed by Western blotting. The functional role of PKCζ was then evaluated in vivo, using intraocular administration of its specific inhibitor. RESULTS: PKCζ was localized in tight junction protein complexes of the retinal pigment epithelium and in photoreceptors inner segments. Strikingly, in outer segment PKCζ staining was restricted to cone photoreceptors. Short-term hyperglycemia induced activation and delocalization of PKCζ from both retinal pigment epithelium junctions and cone outer segment. Outer blood retinal barrier disruption and photoreceptor cone degeneration characterized long-term hyperglycemia. In vivo, reduction of PKCζ overactivation using a specific inhibitor, restored its tight-junction localization and not only improved the outer blood retinal barrier, but also reduced photoreceptor cell-death. CONCLUSIONS: In the retina, hyperglycemia induced overactivation of PKCζ is associated with outer blood retinal barrier breakdown and photoreceptor degeneration. In vivo, short-term inhibition of PKCζ restores the outer barrier structure and reduces photoreceptor cell death, identifying PKCζ as a potential target for early and underestimated diabetes-induced retinal pathology.

Comparison of dengue infection in human mononuclear leukocytes with mosquito C6/36 and mammalian Vero cells using flow cytometry to detect virus antigen

Fluorescent activated cell sorter (FACS) analysis is useful for the detection of cellular surface antigens and intracellular proteins. We used this methodology in order to detect and quantify dengue antigens in highly susceptible cells such as clone C6/36 (Aedes albopictus) and Vero cells (green monkey kidney). Additionally, we analyzed the infection in vitro of human peripheral blood mononuclear leukocytes (PBML). FACS analysis turned out to be a reliable technique to quantify virus growth in traditional cell cultures of C6/36 as well as Vero cells. High rates of infection were achieved with a good statistical correlation between the virus amount used in infection and the percentage of dengue antigen containing cells detected in infected cultures. We also showed that human monocytes (CD14+) are preferred target cells for in vitro dengue infection among PBML. Monocytes were much less susceptible to virus infection than cell lines but they displayed dengue antigens detected by FACS five days after infection. In contrast, lymphocytes showed no differences in their profile for dengue specific immunofluorescence. Without an animal model to reproduce dengue disease, alternative assays have been sought to correlate viral virulence with clinical manifestations and disease severity. Study of in vitro interaction of virus and host cells may highlight this relationship.

An ocellar "pupil" that does not change with light intensity, but with the insect age in Triatoma infestans

The simple eyes (ocelli) of recently emerged adult Triatoma infestans exhibit a narrow elongated "pupil", surrounded by a ring of brown-reddish pigment, the "iris". This pupil does not respond to changes in the illumination, but varies in size after the imaginal ecdysis. This change corresponds, internally, with the growth of the corneal lens and the associated retina up to an age of about 20 days. This has not been previously observed in an insect. The use of this characteristic for recognising young adults of this species is suggested.

Cebus apella (Primata: Cebidae) as a new host for Fonsecalges johnjadini (Acari: Psoroptidae, Cebalginae) with a description of anatomopathological aspects

Mites collected from the auditory canal of Cebus apella (capuchin monkey), family Cebidae, were identified as Fonsecalges johnjadini (Psoroptidae, Cebalginae). It is the first record of this parasite from this monkey. This paper emphasizes the importance of clinical and anatomopathological examinations for parasitic diagnosis in wild animals.