Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane.

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for "dry," or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch's membrane pathology is part of this process. Alterations in human Bruch's membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch's membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch's membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch's membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

Identification of Chlamydia pneumoniae within human choroidal neovascular membranes secondary to age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of blindness in the United States, and increasing evidence suggests that it is an inflammatory disease. The prokaryotic obligate intracellular pathogen Chlamydia pneumoniae is emerging as a novel risk factor in cardiovascular disease, and recent sero-epidemiological data suggest that C. pneumoniae infection is also associated with AMD. In this study, we examined choroidal neovascular membrane (CNV) tissue from patients with neovascular AMD for the presence of C. pneumoniae and determined whether the pathogen can dysregulate the function of key cell types in ways that can cause neovascular AMD. Nine CNV removed from patients with neovascular AMD were examined for the presence of C. pneumoniae by immunohistochemistry (IHC) and polymerase chain reaction (PCR); in addition, we performed PCR on nine non-AMD eyes, and IHC on five non-AMD CNV, seven non-AMD eyes, and one internal limiting membrane specimen. Finally, human monocyte-derived macrophages and retinal pigment epithelial (RPE) cells were exposed to C. pneumoniae and assayed in vitro for the production of pro-angiogenic immunomodulators (VEGF, IL-8, and MCP-1). C. pneumoniae was detected in four of nine AMD CNV by IHC and two of nine AMD CNV by PCR, induced VEGF production by human macrophages, and increased production of IL-8 and MCP-1 by RPE cells. In contrast, none of the 22 non-AMD specimens showed evidence for C. pneumoniae. These data indicate that a pathogen capable of inducing chronic inflammation and pro-angiogenic cytokines can be detected in some AMD CNV, and suggest that infection may contribute to the pathogenesis of AMD.

Endogenous mycotic endophthalmitis: variations in clinical and histopathologic changes in candidiasis compared with aspergillosis.

PURPOSE: To describe clinical and/or histopathologic features that could help distinguish endogenous Candida endophthalmitis from endogenous Aspergillus intraocular inflammation and to provide histologic documentation of intraocular spread of these agents. METHODS: Twenty-five patients who underwent enucleation, 13 with morphologic features and/or positive culture for Aspergillus and 12 with histologic evidence and/or positive culture for Candida were included in the study. Clinical information was sought from each case. Patients with AIDS were excluded. The enucleated globes were analyzed to detect location of the fungi, vascular invasion by these agents, and inflammatory response. RESULTS: Candida endophthalmitis was noted in patients with a history of gastrointestinal surgery, hyperalimentation, or diabetes mellitus, whereas aspergillosis was present in patients who had undergone organ transplantation or cardiac surgery. Histopathologically, the vitreous was the primary focus of infection for Candida, whereas subretinal/subretinal pigment epithelium infection was noted in eyes with aspergillosis. Retinal and choroidal vessel wall invasion by fungal elements was noted in cases of aspergillosis but not in cases with candidiasis. Both infectious agents induced suppurative nongranulomatous inflammation. CONCLUSIONS: Unlike Candida endophthalmitis, aspergillosis clinically presents with extensive areas of deep retinitis/choroiditis, and vitreous biopsy may not yield positive results. Histopathologically, it appears that Aspergillus grows preferentially along subretinal pigment epithelium and subretinal space. This intraocular infection is usually associated with a high rate of mortality caused by cerebral and cardiac complications.

Panophthalmitis due to rhizopus in an AIDS patient: a clinicopathological study.

Various opportunistic infections in the eye have been reported earlier in AIDS. We report a case of panophthalmitis in an AIDS patient where the eviscerated tissue on histopathologic and microbiologic examination showed the fungus Rhizopus.

Altered membrane fatty acids of cultured human retinal pigment epithelium persistently infected with rubella virus may affect secondary cellular function.

Persistent infection with rubella virus (RV) can alter secondary functions of host cells. Previously we had documented defective phagocytosis of latex beads by cultured human retinal pigment epithelial cells (RPE), persistently infected with M-33 RV (RPE/RV). Here, examining possible mechanisms for altered function, we reported significant differences between the total esterified fatty acids (FA) of RPE and RPE/RV membranes, measured by gas liquid chromatography. RPE/RV contained an increased proportion of saturated FA, particularly palmitic acid, with a presence of unusual chromatographic FA peaks co-eluting with odd-numbered long-chain carbon atom FA not normally found in human cells. Apical membrane microvilli, structures essential to phagocytic activity of RPE and RPE/RV, observed by scanning and transmission electron microscopy, were similar in number and appearance between uninfected RPE and RPE/RV cells before and after latex bead addition. However, RPE/RV microvilli, possibly reflecting altered membrane FA composition, engaged latex beads less effectively than uninfected RPE microvilli. In addition, microvilli remained abnormally distributed on RPE/RV cell surfaces at 48 h after latex addition. Thus, RV persistent infection may affect the cellular membrane fluidity and functional activity of human cells with increased saturated FA proportions and altered FA components of membrane phospholipids. These changes may participate in the defective phagocytosis of RPE/RV.

Coronavirus (JHM) replication within the retina: analysis of cell tropism in mouse retinal cell cultures.

The murine coronavirus, mouse hepatitis virus, JHM strain, induces a retinal degenerative disease in adult BALB/c mice. Coronavirus infections are highly species specific with virus exhibiting a strong tissue and cell specificity. In this report we evaluated the cellular basis of JHM virus retinal tropism. Retinal cultures and retinal pigment epithelial (RPE)-retinal mixed cell cultures were prepared from eyes obtained from Balb/c mice. The ability of JHM virus to infect and replicate in these retinal cultures was evaluated by light microscopy, immunofluorescent staining, electron microscopy, and virus isolation. Cytopathology was not observed and virus could not be detected in supernatant fluid in retinal cultures. However, low levels of infectious virus could be detected within the cells for the first 4 days. This observation suggested that cell-to-cell interactions may be critical since virus particles and virus antigens can be seen in vivo within the neural retina and the RPE. In contrast to the retinal cultures, retinal-RPE mixed cultures were supportive to JHM virus replication. Syncytial cytopathology was observed for the first 4 days and virus was isolated from supernatant fluids. By electron microscopy, virus was found intracellularly within vacuoles and extracellularly at the plasma membrane. After Day 4, a persistent virus infection was established in which cells produced virus for 5 weeks without cytopathic effects or cell death. Double-labeling immunofluorescent studies of retinal-RPE mixed cultures showed that the virus antigen was co-expressed with a Muller cell marker, glutamine synthetase. This cell is the most prominent glial element in the retina. These studies demonstrate that JHM virus is capable of establishing a persistent virus infection in mixed retinal (Muller)-RPE cell cultures. Moreover, these data suggest that cell-to-cell interactions influence the establishment of coronavirus infections in the retina.

The retinal pigment epithelium: a versatile partner in vision.

The retinal pigment epithelium (RPE) is a monolayer of cuboidal cells that lies in close association with the rod and cone photoreceptors. This epithelium has diverse features, three of which are discussed in some detail in this review, namely the daily phagocytosis of rod and cone outer segment fragments that are shed from their distal ends; the uptake, processing, transport and release of vitamin A (retinol) and some of its visual cycle intermediates (retinoids); and some of the aspects of its apical and basolateral membrane polarity that are the reverse of most other epithelia. Phagocytosis takes place at the apical surface via membrane receptor-mediated processes that are not yet well defined. Retinol uptake occurs at both the basolateral and apical surfaces by what appear to be separate receptor-mediated processes. The release of a crucial retinoid, 11-cis retinaldehyde (11-cis retinal), occurs solely across the apical membrane. Delivery of retinol across the basolateral membrane is mediated by a retinol binding protein (RBP) that is secreted by the liver as a complex with retinol (vitamin A). Within the cell, retinol and its derivatives are solubilized by intracellular retinoid binding proteins that are selective for retinol (cellular retinol binding protein, CRBP) and 11-cis retinoids (cellular retinal binding protein, CRALBP). Release of 11-cis retinal across the apical membrane and re-uptake of retinol from the photoreceptors during the visual cycle is promoted by an intercellular retinoid binding protein (IRBP). Na,K-ATPase, the membrane-integrated enzyme required to set up the ion gradients that drive other ion transporters, is largely localized to the apical membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Immortalization of polarized rat retinal pigment epithelium.

Rat retinal pigment epithelial (RPE) cells were immortalized by infection with a temperature-sensitive tsA SV40 virus and following cloning and selection for epithelial properties the polarized RPE-J cell line was obtained. At the permissive temperature of 33 degrees C, RPE-J cells behave as an immortalized cell line. When RPE-J cells are grown on nitrocellulose filters coated with a thin layer of Matrigel in the presence of 10(-8) M retinoic acid for 6 days at 33 degrees C and then switched for 33-36 hours to the non-permissive temperature of 40 degrees C, they acquire a differentiated polarized RPE phenotype. Under these growth conditions, RPE-J cells exhibit circumferential staining for the tight-junction protein ZO-1 and acquire a transepithelial resistance of 350 ohms cm2. Morphologically, RPE-J cells exhibit a characteristic RPE morphology with extensive apical microvilli as well as numerous dense bodies including premelanosomes and varied multilamellar structures. Ruthenium red labeling revealed the frequent basal localization of the tight junction. The cells were identified to be of rat RPE origin by their expression of the rat RPE marker RET-PE2 and their ability to phagocytose latex beads. While RPE-J cells are capable of sorting influenza and vesicular stomatitis virus to the apical and basal surfaces, respectively, the Na,K-ATPase is not polarized and the neural cell adhesion molecule, N-CAM, is localized exclusively to the lateral surface. In vivo the apical surface of RPE interacts with the adjacent neural retina and the Na,K-ATPase and N-CAM are both apical; the altered polarity of these two proteins in RPE-J cells may be a consequence of the absence of apical interaction with the neural retina in culture. Previous studies of RPE have been restricted to the use of primary cultures and the RPE-J cell line should prove an excellent model system for the study of the mechanisms determining the characteristic polarity and functions of the retinal pigment epithelium.

Polarized budding of vesicular stomatitis and influenza virus from cultured human and bovine retinal pigment epithelium.

The retinal pigment epithelium (RPE) is able to perform a variety of functions because of its high degree of plasma membrane polarity. Some aspects of this polarity such as the localization of the majority of Na-K ATPase to the apical membrane distinguish the RPE from kidney cells and most other transporting epithelia. The polarized budding of enveloped viruses such as vesicular stomatitis and influenza from the basolateral and apical membrane, respectively, has been used to study mechanisms underlying the domain-specific sorting of membrane proteins in cultured epithelial cell lines. These processes also serve as a useful index of the degree of polarization in epithelial cell cultures. Viral budding from apical and basolateral RPE membranes was used in this study to determine whether the sorting of viral envelope membrane proteins by the RPE is reversed in polarity from that of kidney cells and, if so, whether this might predict a fundamental difference in membrane protein sorting for RPE. The results clearly indicate that the polarity of viral membrane sorting and subsequent viral budding is the same in RPE as in other polarized epithelial cell lines examined to date.

Elevated intraocular pressure, pigment dispersion and dark hypopyon in endogenous endophthalmitis from Listeria monocytogenes.

Listeria monocytogenes endophthalmitis occurred in an immunologically competent patient with no identifiable extraocular septic focus. The patient presented with a dark hypopyon and markedly elevated intraocular pressure, and the diagnosis was established by culture and histopathologic examination of ocular fluid. Four of the fourteen reported cases of Listeria monocytogenes endophthalmitis also presented with a dark hypopyon, and all cases had markedly elevated intraocular pressure. The presence of a dark hypopyon and elevated intraocular pressure may indicate endogenous intraocular infection with Listeria monocytogenes, even in an apparently healthy host.

Histopathological studies of a case of cytomegalovirus retinochoroiditis.

A case of cytomegalovirus (CMV) retinochoroiditis initially misdiagnosed as fungal endophthalmitis is reported. An 83-year-old man who was suspected of having cholangiocarcinoma presented uveitis in both eyes. Candida endophthalmitis was suspected on the basis of ophthalmic findings and past history, which included systemic corticosteroid administration and intravenous hyperalimentation. Intravenous treatment with miconazole was not effective. At autopsy, 3 months after the initial ophthalmological examination, the right eye was enucleated and examined histologically and histochemically. Light microscopic examination showed extensive retinal necrosis and numerous cytomegalic cells, so-called owl's eye cells, with intranuclear and intracytoplasmic inclusion bodies. CMV particles were seen by electron microscopy, and CMV-infected cells were observed by immunohistochemical staining by the direct method with fluorescein-labeled antibodies. These findings indicate that in suspected cases of fungal endophthalmitis various tests should also be carried out for CMV.

Cytomegalovirus replication in cultured human retinal pigment epithelial cells.

Retinal pigment epithelium (RPE) was isolated from the globe of a donor positive for human immunodeficiency virus (HIV) who had cytomegalovirus (CMV) retinitis secondary to acquired immunodeficiency syndrome (AIDS). In culture, the cells exhibited normal epithelioid morphology by phase contrast microscopy. After two weeks the cells developed cytomegaly and dense intranuclear and cytoplasmic inclusions and, eventually, died. Transmission electron microscopy (EM) demonstrated intranuclear nonenveloped virus particles 80-120 nm in diameter consistent with a herpes type infection. Immunofluorescence staining demonstrated the presence of CMV antigens. Conditioned medium from the infected cells caused infection in RPE cells isolated from normal donors. Hybridization assay demonstrated the presence of CMV DNA and indicated that the time course of the infection was similar, but not identical to infection in MRC-5 and HEL cells. We conclude that cultured human RPE is a permissive host for CMV.

Replication of HIV in human fetal retinal cultures and established pigment epithelial cell lines.

The ability of the human immunodeficiency virus type 1 (HIV-1) to replicate in cells derived from ocular tissue was studied. Primary retinal cultures (containing both glial and neuronal cells) were found to support the replication of HIV upon transfection with molecularly cloned proviral DNA. In addition, established retinal pigment epithelial (RPE) cell lines also produced HIV particles upon transfection. HIV released by these cell lines was able to infect and induce characteristic cytopathic effects in T4+ cells. An indicator plasmid containing the HIV long terminal repeat sequences (LTR) linked to the chloramphenicol acetyltransferase gene showed barely detectable activity in RPE cells and was transactivated by the addition of the HIV "tat" gene. Based on these observations, direct infection of ocular tissue derived cells such as RPE, fetal retinal cells, retinoblastoma cells (Y 79, WER1), choroidal endothelial cells (Chor 55) (mix culture) and corneal fibroblasts (K61) by HIV was attempted. HIV replication in these cells was not detected by reverse transcriptase, antigen and transactivation function assays.

Chorioretinal disease patterns in congenic mice following intraocular inoculation with HSV-1.

Disease patterns and immunologic parameters were studied employing inbred and Igh-1 disparate congenic mice to determine the role of host genetics and Igh-1-linked gene products in the von Szily model of viral chorioretinitis. Following intracameral inoculation of 1.5 x 10(4) PFU HSV-1 (KOS), 100% of BALB/c (Igh-1a), 62% of A/J (Igh-1e) and none of the C57BL/6J (Igh-1b) inbred mice developed contralateral necrotizing chorioretinitis. Multigenic differences between inbred mice prohibit conclusions about the specific role of Igh-1-linked immune regulation in this model. In order to more exactly define Igh-1-specific restriction of HSV-1-mediated chorioretinitis, Igh-1-disparate, congenic BALB/c mice were studied following both anterior chamber and intravitreal inoculation protocols. Anterior chamber inoculation resulted in contralateral retinal necrosis in 75% of BALB/c (Igh-1a) mice, 30% of C.AL-20 (Igh-1d) and 5% of the C.B-17 (Igh-1b) congenic mice; all strains showed ipsilateral retinal sparing. Following intravitreal inoculation of HSV-1 a similar restricted disease pattern was found in contralateral eyes. Contralateral chorioretinitis developed in 30% of BALB/c, 15% of C.AL-20 and 6% of C.B-17 mice. Ipsilateral disease, however, was found in all murine strains. These disease patterns developed despite equivalent suppression of systemic DTH and equivalent RPE permissivity to viral replication. These data demonstrate that host genetics strongly regulates contralateral HSV-1-mediated chorioretinal disease patterns by a mechanism unrelated to the development of systemic suppression of DTH and specifically support a dominant role for gene products linked to the Igh-1 locus in the immunomodulation of ocular disease.

Demonstration of cytomegalovirus retinitis by in situ DNA hybridization.

Five cases of the acquired immune deficiency syndrome (AIDS), each exhibiting retinitis, were studied by DNA hybridization technique to detect viral infection. Five involved eyes were obtained at the time of autopsy from three patients who had received no treatment for the retinitis. Retinal biopsy specimens were obtained at the time of surgery from two other patients who developed retinal detachments after being treated with ganciclovir (dihydroxy propoxymethyl guanine). DNA hybridization revealed cytomegalovirus in retina and retinal pigment epithelium in all five specimens from patients who had not been treated with ganciclovir. No hybridization occurred in the two retinal biopsy specimens obtained from the ganciclovir-treated patients. These results suggest that in situ DNA hybridization is a highly specific and easily interpretable means of establishing the tissue diagnosis of viral retinitis.

Differential sensitivity of cultured retinal neurons and photoreceptors to herpes simplex infection.

Infection of the retina with herpes simplex virus type 1 (HSV-1) causes devastating lesions usually leading to blindness. However, the interactions between individual retinal cell types and this virus have not been well characterized, probably because of limitations posed by the complexity of the intact retina. We have now approached this problem through the use of separate, purified populations of isolated chick embryo retinal neurons and photoreceptor cells, of glial cells, and of pigmented epithelial cells. This manuscript deals with the initial part of these studies, aimed at determining the susceptibility of different retinal types to HSV-1 infection. The different cultures were exposed to HSV-1 for 3-48 hr, and cell infection was evaluated by immunocytochemical detection of viral antigens or by autoradiographic study of viral DNA replication. Practically 100% of the retinal glial cells and pigmented epithelial cells appeared susceptible to HSV-1 infection. On the other hand, as many as 70% of the neurons present in glia-free, pigment epithelium-free cultures, also appeared infected after a 24-hr exposure to the virus. Neuronal susceptibility to HSV-1 was already present in early (2-day) cultures, was time- and concentration-dependent, and led to neuronal degeneration after 24-48 hr. Neuronal infection was also corroborated by the detection of viral particles by transmission electron microscopy. Photoreceptor cells were consistently and selectively resistant to HSV-1 infection at all the concentrations and time points investigated. Both immunocytochemical and autoradiographic studies showed similar results. Photoreceptor resistance to HSV-1 appears to be selective, since they could be readily infected with RNA viruses such as vesicular stomatitis virus and influenza virus. These cell culture preparations offer an attractive system for the investigation of cellular mechanisms involved in the differential susceptibility of retinal cells to viral infection. Moreover, they could also help in the screening of treatments potentially capable of preventing and (or) curing HSV-induced retinal infection.