Mark A. Fields

Neutrophils protect the retina of the injected eye from infection after anterior chamber inoculation of HSV-1 in BALB/c mice.

PURPOSE To determine whether infiltrating polymorphonuclear leukocytes PMNs play a role in preventing early direct anterior-to-posterior spread of herpes simplex virus (HSV)-1 and/or in preventing the spread of HSV-1 from the brain back to the retina of the injected eye after anterior chamber (AC) inoculation. METHODS BALB/c mice were treated with monoclonal antibody RB6-8C5 (Gr-1) against PMNs or control IgG and inoculated with HSV-1. RESULTS In Gr-1-treated mice, PMNs were depleted in the peripheral blood and in the HSV-1-infected eye. More virus (2-3 logs) was recovered from the inoculated eye of Gr-1 antibody-treated mice than from control mice. Immunohistochemistry revealed disseminated virus-infected cells in the junction between the anterior and the posterior segment and also in the posterior segment of the HSV-1-inoculated eye in Gr-1-treated mice. In control IgG-treated mice, virus-infected cells were observed only within the AC. More virus (3 logs) was recovered from the contralateral suprachiasmatic nucleus (SCN), and increased virus staining was observed in the ipsilateral optic nerve of Gr-1-treated mice compared with control mice. In Gr-1-treated mice, the central retina was virus-infected in a patchy fashion beginning on day 7 post infection (pi), and the infection progressed to involve the entire retina. CONCLUSIONS Since both direct anterior-to-posterior spread of virus and spread via the optic nerve occurred in PMN-depleted mice, these results suggest that PMNs play an important role both in limiting intraocular spread of virus in the injected eye and in controlling spread of the virus from the brain into the optic nerve and retina of the injected eye.

Potential of Induced Pluripotent Stem Cells (iPSCs) for Treating Age-Related Macular Degeneration (AMD)

The field of stem cell biology has rapidly evolved in the last few decades. In the area of regenerative medicine, clinical applications using stem cells hold the potential to be a powerful tool in the treatment of a wide variety of diseases, in particular, disorders of the eye. Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are promising technologies that can potentially provide an unlimited source of cells for cell replacement therapy in the treatment of retinal degenerative disorders such as age-related macular degeneration (AMD), Stargardt disease, and other disorders. ESCs and iPSCs have been used to generate retinal pigment epithelium (RPE) cells and their functional behavior has been tested in vitro and in vivo in animal models. Additionally, iPSC-derived RPE cells provide an autologous source of cells for therapeutic use, as well as allow for novel approaches in disease modeling and drug development platforms. Clinical trials are currently testing the safety and efficacy of these cells in patients with AMD. In this review, the current status of iPSC disease modeling of AMD is discussed, as well as the challenges and potential of this technology as a viable option for cell replacement therapy in retinal degeneration.

Tumor necrosis factor alpha and macrophages in the brain of herpes simplex virus type 1-infected BALB/c mice

After uniocular anterior chamber (AC) inoculation of herpes simplex virus type 1 (HSV-1), virus and TNF alpha (TNF-α) are detected in the suprachiasmatic nuclei (SCN). The goal of this study was to investigate the role of TNF-α and macrophages in the brain of HSV-1-infected BALB/c mice. Mice were treated with thalidomide for TNF-α inhibition or injected with clodronate liposomes to deplete macrophages, and the AC of one eye (ipsilateral) was injected with HSV-1 (KOS). The location of HSV-1, macrophages, and TNF-α was determined by fluorescence immunohistochemistry and the titer of virus was determined by plaque assay. Inhibition of TNF-α was determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and depletion of macrophages was assessed by flow cytometry. In thalidomide-treated mice, TNF-α RNA levels were reduced in the SCN. Both SCN were infected by day 5 post inoculation (p.i.) and the titer of virus in the SCN contralateral to the side of injection was increased. The number of splenic macrophages was significantly reduced in clodronate-treated mice compared with controls. In macrophage-depleted mice, both SCN were infected at day 6 p.i. and the titer of virus in the SCN of these mice was increased at days 6 and 7 p.i. compared with controls. The titer of virus in the contralateral (uninoculated) eye of macrophage-depleted mice was increased at day 7 p.i. Fewer F4/80+ cells were observed in the SCN of macrophage-depleted mice. The results of these studies suggest that TNF-α plays a role in limiting virus replication in the SCN of euthymic BALB/c mice and that one source of TNF-α is macrophages.

Infiltrating Cells and IFNγ Production in the Injected Eye after Uniocular Anterior Chamber Inoculation of HSV-1

PURPOSE After uniocular anterior chamber (AC) inoculation with HSV-1, the anterior segment of the injected eye becomes inflamed and infected; however, virus does not spread from the anterior segment and infect the retina of the injected eye. The purpose of this study was to identify early infiltrating cells and to determine whether infiltrating cells produce interferon (IFN)gamma. METHODS Euthymic, female, BALB/c mice were injected in one AC with 3 x 10(4) PFU of HSV-1 (KOS) in a volume of 2 microL. Mice from each group were killed at 12, 24, 36, 48, and 72 hours post injection (pi), the eyes were enucleated, and frozen sections were stained with antibodies specific for IFNgamma, Mac-1 (CD11b), CD49b, F4/80, CD4, CD8, and CD11c. The same antibodies were also used to stain single-cell suspensions of ocular cells for flow cytometry. RESULTS In the anterior segment of the injected eye, the ciliary body, and iris were virus infected and inflamed, and infiltrating cells increased throughout the period of observation. Mac-1(+), CD49b(+), and F4/80(+) cells colocalized with IFNgamma in the anterior segment as early as 12 hours pi, and the percentage of Mac-1(+) cells increased in the injected eye beginning at 24 hours pi and continued to 72 hours pi. CONCLUSIONS Taken together, these results demonstrate that Mac-1(+) cells are important IFNgamma-producing cells in the injected eye before day 3 and suggest that the IFNgamma produced by these cells is involved in inhibition of anterior to posterior spread of virus in the injected eye.

Uniocular Anterior Chamber Inoculation of a Tumor Necrosis Factor Alpha-Expressing Recombinant of Herpes Simplex Virus Type 1 Results in More Rapid Destruction and Increased Viral Replication in the Retina of the Uninoculated Eye

ABSTRACT Tumor necrosis factor alpha (TNF-α) has been shown to have a protective role in the eyes and brains of herpes simplex virus type 1 (HSV-1)-infected mice. To determine whether overexpression of TNF-α affected the course of virus infection following uniocular anterior chamber inoculation, a recombinant of HSV-1 that produces TNF-α constitutively (KOSTNF) was constructed. BALB/c mice were injected with the TNF-α recombinant, a recombinant containing the pCI plasmid, a recombinant rescue virus, or the parental virus. Flow cytometry and immunohistochemistry were used to identify virus-infected cells and to determine the numbers and types of infiltrating inflammatory cells in the uninjected eyes. Virus titers were determined by plaque assay. There were no differences among the groups in virus titers or the route and timing of virus spread in the injected eyes or in the suprachiasmatic nuclei. However, in the uninjected eyes of KOSTNF-infected mice, TNF-α expression was increased and there were more viral antigen-positive cells and immune inflammatory cells. There was earlier microscopic evidence of retinal infection and destruction in these mice, and the titers of virus in the uninjected eyes were significantly increased in KOSTNF-infected mice on day 7 postinfection compared with those of KOSpCI-, KOS6βrescue-, or KOS6β-infected mice. The results suggest that instead of moderating infection and reducing virus spread, overexpression of TNF-α has deleterious effects due to increased inflammation and virus infection that result in earlier destruction of the retina of the uninoculated eye.

Extracellular matrix nitration alters growth factor release and activates bioactive complement in human retinal pigment epithelial cells

Purpose We have shown previously that non-enzymatic nitration (NEN) of the extracellular matrix (ECM), which serves as a model of Bruch’s membrane (BM) aging, has a profound effect on the behavior of the overlying retinal pigment epithelial (RPE) cells, including altered phagocytic ability, reduced cell adhesion, and inhibition of proliferation. We know that transplanted RPE monolayers will encounter a hostile sub-RPE environment, including age-related alterations in BM that may compromise cell function and survival. Here we use our previous NEN model of BM aging to determine the effects of NEN of the ECM on growth factor release and complement activation in RPE cells. Methods Human induced-pluripotent stem cells (iPSCs) were differentiated into RPE cells, and confirmed by immunohistochemistry, confocal microscopy, and polymerase chain reaction. IPSC-derived RPE cells were plated onto RPE-derived ECM under untreated or nitrite-modified conditions. Cells were cultured for 7 days and barrier function measured by transepithelial resistance (TER). Vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), and complement component C3a were measured using enzyme-linked immunosorbent assay (ELISA). Results On average nitrite-modified ECM increased VEGF release both apically and basally by 0.15 ± 0.014 ng/mL (p <0.0001) and 0.21 ± 0.022 ng/mL (p <0.0001), respectively, in iPSC-derived RPE cells. Nitrite-modified ECM increased PEDF release in iPSC-derived RPE cells apically by 0.16 ± 0.031 ng/mL (p <0.0001), but not basally (0.27 ± 0.015 vs. 0.32 ± 0.029 ng/mL, (p >0.05)). Nitrite-modified ECM increased production of C3a in iPSC-derived RPE cells by 0.52 ± 0.123 ng/mL (p <0.05). Conclusion Nitrite-modified ECM increased VEGF, PEDF release, and C3a production in human iPSC-derived RPE cells. This model demonstrates changes seen in the basement membrane can lead to alterations in the cell biology of the RPE cells that may be related to the development of age-related macular degeneration.