Corneal Nerve Ablation Abolishes Ocular Immune Privilege by Downregulating CD103 on T Regulatory Cells.

Purpose: Severing corneal nerves during orthotopic corneal transplantation elicits the elaboration of the neuropeptide substance P (SP), which induces the generation of CD11c+ contrasuppressor (CS) cells. CS cells disable T regulatory cells (Tregs) that are induced when antigens enter the anterior chamber (AC), either by direct injection or by orthotopic corneal transplantation. This study examined the crucial cell surface molecules on Tregs that are adversely affected by CS cells that are generated by severing corneal nerves. Methods: CS cells were induced by producing shallow 2.0-mm circular incisions in the corneal epithelium in BALB/c mice. CD8+ Tregs were generated by injecting ovalbumin into the AC. The effects of CS cells and SP on the expression and function of two cell surface molecules (CD103 and the receptor of interferon-γ) that are crucial for the induction and function of CD8+ Tregs were analyzed. Results: SP converted CD11c+, but not CD11c- , dendritic cells (DCs) to CS cells. Severing corneal nerves resulted in a 66% reduction in the expression of CD103 on CD8+ AC-associated immune deviation (ACAID) Tregs, and a 50% reduction in the interferon-γ receptor (IFN-γR). These effects could be mimicked in vitro by coculturing CS cells with CD8+ ACAID Tregs. Conclusions: The elaboration of SP in response to corneal nerve ablation converts CD11c+ DCs to CS cells. CS cells disable CD8+ ACAID Tregs by downregulating two crucial cell surface molecules, CD103 and IFN-γR, by an SP-dependent pathway. Blocking this pathway may provide a means of restoring ocular immune privilege in corneas subjected to corneal nerve injury.

Defective FasL expression is associated with increased resistance to melanoma liver metastases and enhanced natural killer cell activity.

The objective was to determine if the absence of FasL signaling would affect melanoma liver metastases by influencing the antimelanoma properties of liver natural killer (NK) cells. Melanoma liver metastases were induced in wild-type C57BL/6 mice and the gld/gld mutant C57BL/6 mouse strain that expresses a defective form of FasL (CD95L) that fails to engage and signal via the Fas receptor (CD95). Liver metastases were produced by intrasplenic injection of B16LS9 melanoma cells. Liver NK cell activity directed against murine B16LS9 melanoma cells was determined in a 24 h in-vitro cytotoxicity assay. Liver NK cells, NK T cells, and the NK cell surface activation marker, NKG2D, were measured by flow cytometry. Mice expressing defective FasL displayed reduced, rather than enhanced, melanoma liver metastases that coincided with increased liver NK cell-mediated tumor cell cytotoxicity. Enhanced cytotoxicity was not mediated by perforin, tumor necrosis factor-α, or tumor necrosis-associated apoptosis-inducing ligand but was closely associated with elevated interferon-γ in the tumor-bearing liver. FasL-defective gld/gld mice also displayed reduced numbers of liver NK T cells, which have been previously implicated in suppression on liver NK cell activity. The absence of functional FasL in the liver correlates with a heightened, not diminished, resistance to melanoma liver metastases. The resistance to liver metastases coincides with a significant, albeit transient, increase in liver NK cytotoxicity and elevated levels of interferon-γ in the liver.

Schnyder corneal dystrophy-associated UBIAD1 inhibits ER-associated degradation of HMG CoA reductase in mice.

Autosomal-dominant Schnyder corneal dystrophy (SCD) is characterized by corneal opacification owing to overaccumulation of cholesterol. SCD is caused by mutations in UBIAD1, which utilizes geranylgeranyl pyrophosphate (GGpp) to synthesize vitamin K2. Using cultured cells, we previously showed that sterols trigger binding of UBIAD1 to the cholesterol biosynthetic enzyme HMG CoA reductase (HMGCR), thereby inhibiting its endoplasmic reticulum (ER)-associated degradation (ERAD) (Schumacher et al. 2015). GGpp triggers release of UBIAD1 from HMGCR, allowing maximal ERAD and ER-to-Golgi transport of UBIAD1. SCD-associated UBIAD1 resists GGpp-induced release and is sequestered in ER to inhibit ERAD. We now report knockin mice expressing SCD-associated UBIAD1 accumulate HMGCR in several tissues resulting from ER sequestration of mutant UBIAD1 and inhibition of HMGCR ERAD. Corneas from aged knockin mice exhibit signs of opacification and sterol overaccumulation. These results establish the physiological significance of UBIAD1 in cholesterol homeostasis and indicate inhibition of HMGCR ERAD contributes to SCD pathogenesis.

Induction of Contrasuppressor Cells and Loss of Immune Privilege Produced by Corneal Nerve Ablation.

Purpose: Severing of corneal nerves in preparation of corneal transplantation abolishes immune privilege of subsequent corneal transplants placed into either eye: a phenomenon termed sympathetic loss of immune privilege (SLIP). SLIP is due to the disabling of T regulatory cells (Tregs) by CD11c+ contrasuppressor (CS) cells. This study characterized the induction, function, and manipulation of CS cell activity and the effect of these cells on Tregs induced by anterior chamber-associated immune deviation (ACAID). Methods: CS cells were induced using a 2.0-mm trephine to score the corneal epithelium. CD11c+ CS cells were evaluated by adoptive transfer and by their capacity to disable CD8+ ACAID Tregs in local adoptive transfer (LAT) of suppression assays. CD11c+ cells were deleted from the ocular surface by subconjunctival injection of clodronate-containing liposomes. Results: CD11c+ CS cell were radiosenstive and long lived. As few as 1000 CS cells blocked the suppressive activity of previously generated CD8+ ACAID Tregs, indicating that CS cells act at the efferent arm of the immune response. Depletion of resident CD11c+ cells at the ocular surface prevented the generation of CS cells. Conclusions: Corneal nerve injury that occurs during keratoplasty converts ocular surface CD11c+ cells into CS cells that block CD8+ Tregs, which are induced by introducing antigens into the anterior chamber (i.e., ACAID Tregs). Depletion of CD11c+ cells at the ocular surface prevents the generation of CS cells and may be a useful strategy for preventing SLIP and enhancing the survival of second corneal transplants.

Pathobiology and Immunobiology of Acanthamoeba Keratitis: Insights from Animal Models
.

Acanthamoeba keratitis (AK) is a rare but sight-threatening disease caused by pathogenic species of Acanthamoeba. Despite its ubiquitous nature, the incidence of AK is relatively low compared to other forms of infectious keratitis. Although contact lens wear is a major risk factor, exposure to contaminated water and ocular trauma are also associated with AK. Once a patient develops AK the prognosis is very poor unless an aggressive treatment regimen is initiated early. Some of the intriguing features of AK are the lack of immunological memory, resistance of the dormant cyst form to treatment, differences between the pathogenic strains and soil isolates of Acanthamoeba and the unique role of the innate immune system in controlling this disease. Understanding the series of steps involved in the pathogenesis of the disease and the host immune response against Acanthamoeba antigens is crucial for developing effective therapeutic strategies targeting the disease.

Effect of Corneal Nerve Ablation on Immune Tolerance Induced by Corneal Allografts, Oral Immunization, or Anterior Chamber Injection of Antigens.

Purpose: Severing corneal nerves during corneal transplantation does not affect first corneal transplants, but abolishes immune privilege of subsequent corneal allografts. This abrogation of immune privilege is attributable to the disabling of T regulatory cells (T regs) induced by corneal transplantation. The goal of this study was to determine if severing corneal nerves induces the development of contrasuppressor (CS) cells, which disable T regs that impair other forms of immune tolerance. Methods: Effect of corneal nerve ablation on immune tolerance was assessed in four forms of immune tolerance: anterior chamber-associated immune deviation (ACAID); oral tolerance; corneal transplantation, and intravenously (IV) induced immune tolerance. T regulatory cell activity was assessed by adoptive transfer and by local adoptive transfer (LAT) of suppression assays. Results: Corneal nerve ablation prevented ACAID and oral tolerance, but did not affect IV-induced immune tolerance. Contrasuppressor cells blocked the action of T regs that were generated by anterior chamber injection, oral tolerance, or orthotopic corneal transplantation. The neuropeptide substance P (SP) was crucial for contrasuppressor activity as CS cells could not be induced in SP-/- mice and the SP receptor inhibitor, Spantide II, prevented the expression of CS cell activity in vivo. Contrasuppressor cells expressed CD11c surface marker that identifies dendritic cells (DC). Conclusions: The loss of immune privilege produced by corneal nerve ablation following corneal transplantation extends beyond the eye and also affects immune tolerance induced through mucosal surfaces and appears to be mediated by a novel cell population of CD11c+ CS cells that disables T regs.

LRBA is Essential for Allogeneic Responses in Bone Marrow Transplantation.

The PH-BEACH-WD40 (PBW) protein family members play a role in coordinating receptor signaling and intracellular vesicle trafficking. LPS-Responsive-Beige-like Anchor (LRBA) is a PBW protein whose immune function remains elusive. Here we show that LRBA-null mice are viable, but exhibit compromised rejection of allogeneic, xenogeneic and missing self bone-marrow grafts. Further, we demonstrate that LRBA-null Natural Killer (NK) cells exhibit impaired signaling by the key NK activating receptors, NKp46 and NKG2D. However, induction of IFN-γ by cytokines remains intact, indicating LRBA selectively facilitates signals by receptors for ligands expressed on the surface of NK targets. Surprisingly, LRBA limits immunoregulatory cell numbers in tissues where GvHD is primed or initiated, and consistent with this LRBA-null mice also demonstrate resistance to lethal GvHD. These findings demonstrate that LRBA is redundant for host longevity while being essential for both host and donor-mediated immune responses and thus represents a unique and novel molecular target in transplant immunology.

Inhibition of hypoxia inducible factor-1α downregulates the expression of epithelial to mesenchymal transition early marker proteins without undermining cell survival in hypoxic lens epithelial cells.

PURPOSE: The purpose of this study was to identify potential therapeutic strategies to slow down or prevent the expression of early-onset epithelial to mesenchymal transition (EMT) marker proteins (fibronectin and alpha smooth muscle actin, α-SMA) without sacrificing the synthesis and accumulation of the prosurvival protein vascular endothelial growth factor (VEGF) in cultured virally transformed human lens epithelial (HLE) cells. METHODS: HLE-B3 cells, maintained in a continuous hypoxic environment (1% oxygen), were treated with SB216763, a specific inhibitor of glycogen synthase kinase-3ß (GSK-3ß) catalytic activity. Western blot analysis was employed to detect the cytoplasmic and nuclear levels of ß-catenin, as well as the total lysate content of fibronectin and α-SMA. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of VEGF in cell culture medium. A hypoxia-inducible factor-1α (HIF-1α) translation inhibitor and an HIF-2α translation inhibitor were independently employed to evaluate the effect of hypoxia inducible factor inhibition on EMT marker protein and VEGF expression. XAV932 was used to assess the suppression of nuclear ß-catenin and its downstream effect on EMT marker proteins and VEGF expression. RESULTS: SB216763-treated HLE-B3 cells caused marked inhibition of GSK-3ß activity prompting a significant increase in the translocation of cytoplasmic ß-catenin to the nucleus. The enhancement of nuclear ß-catenin looked as if it positively correlated with a significant increase in the basal expression of VEGF as well as increased expression of fibronectin and α-SMA. In conjunction with SB216763, coadministration of an HIF-1α translation inhibitor, but not an HIF-2α translation inhibitor, markedly suppressed the expression of fibronectin and α-SMA without affecting VEGF levels. Treatment with XAV932 significantly reduced the level of nuclear ß-catenin, but the levels of neither the EMT marker proteins nor VEGF were changed. CONCLUSIONS: Recently, we reported that nuclear ß-catenin, but not HIF-2α, regulates the expression of fibronectin and α-SMA in atmospheric oxygen. In marked contrast, data from the hypoxic condition clearly establish that nuclear ß-catenin plays little apparent role in the expression of EMT marker proteins. Instead, the loss of HIF-1α (but not HIF-2α) decreases the expression of the EMT marker proteins without sacrificing the levels of the prosurvival protein VEGF. These findings support the development of a potentially relevant therapeutic strategy to undermine the progression of normal cells to the mesenchymal phenotype in the naturally hypoxic lens without subverting cell viability.

TCF4 Triplet Repeat Expansion and Nuclear RNA Foci in Fuchs' Endothelial Corneal Dystrophy.

PURPOSE: Expansion of the intronic CTG18.1 triplet repeat locus within TCF4 contributes significant risk to the development of Fuchs' endothelial corneal dystrophy (FECD) in Eurasian populations, but the mechanisms by which the expanded repeats result in degeneration of the endothelium have been hitherto unknown. The purpose of this study was to examine FECD endothelial samples for the presence of RNA nuclear foci, the hallmark of toxic RNA, as well as evidence of haploinsufficiency of TCF4. METHODS: Using fluorescence in situ hybridization, we examined for the presence of nuclear RNA foci containing expanded CUG transcripts in corneal endothelial samples from FECD subjects with CTG18.1 expansion. We also examined for any changes in expression levels of TCF4 by quantitative real-time PCR. RESULTS: Numerous discrete nuclear RNA foci were identified in endothelial samples of FECD subjects (n = 8) harboring the CTG18.1 expansion, but not in controls lacking the expansion (n = 5) (P = 7.8 × 10(-4)). Percentage of cells with foci in expansion-positive endothelial samples ranged from 33% to 88%. RNA foci were absent in endothelial samples from an FECD subject without CTG18.1 expansion and a subject with endothelial dysfunction without FECD. Expression of the constitutive TCF4 exon encoding the basic helix-loop-helix domain was unaltered with CTG18.1 expansion. CONCLUSIONS: Our findings suggest that the RNA nuclear foci are pathognomonic for CTG18.1 expansion-mediated endothelial disease. The RNA nuclear foci have been previously found only in rare neurodegenerative disorders caused by repeat expansions. Our detection of abundant ribonuclear foci in FECD implicates a role for toxic RNA in this common disease.

Lenticular cytoprotection, part 2: link between glycogen synthase kinase-3ß, epithelial to mesenchymal transition, and mitochondrial depolarization.

PURPOSE: The inhibition of GSK-3ß blocks mitochondrial membrane permeability transition (mMPT) for HLE-B3 cells in atmospheric oxygen. GSK-3ß, as part of a multifactorial complex, also regulates nuclear levels of ß-catenin, a known coordinator of cell survival and adhesion. The purpose of these studies was to demonstrate a novel, but likely disadvantageous, link between ß-catenin's influence on the expression of the pro-survival protein, vascular endothelial growth factor (VEGF), resulting in enhanced lens epithelial cell mitochondrial protection against depolarization and nuclear ß-catenin as an inducer of epithelial to mesenchymal transition (EMT). METHODS: Virally transformed human lens epithelial cells (HLE-B3) were treated with SB216763, a specific inhibitor of GSK-3ß catalytic activity and XAV939, a specific ß-catenin inhibitor that bars the translocation of ß-catenin from cytoplasm to the nucleus. Western blot analysis was employed to detect the levels of cytoplasmic and nuclear ß-catenin and phospho-ß-catenin, pBcl-2 and the EMT proteins, α-smooth muscle actin (α-SMA), and fibronectin. ELISA was used to measure the levels of VEGF in cell culture supernatants. JC-1 analysis was performed to analyze the influence of either SB216763 or XAV939 on mitochondrial depolarization. RESULTS: Cultured lens epithelial cells maintained in hypoxia (1% oxygen) and subsequently reintroduced into atmospheric oxygen and treated with the GSK-3ß inhibitor SB216763 illustrated a marked inhibition of phosphorylation of glycogen synthase (downstream substrate of GSK-3ß) and significant increase in nuclear translocation of ß-catenin. The augmented nuclear ß-catenin levels positively correlated with increased expression of α-SMA and fibronectin, both marker proteins indicative of EMT. The enhanced nuclear ß-catenin activity also elicited increased VEGF and pBcl-2 expression, resulting in increased resistance to mitochondrial depolarization. Treatment of the cells with the ß-catenin inhibitor XAV939 resulted in decreased expression of nuclear ß-catenin, VEGF levels, pBcl-2, and EMT proteins, as well as increased mitochondrial depolarization. CONCLUSIONS: The data support a model whereby the onset of epithelial to mesenchymal transition may circuitously benefit from the enhanced synthesis of VEGF by setting up a potentially harmful situation whereby the resulting mesenchymal cell population may be more resistant to mitochondrial depolarization than the lens epithelial cell population from which it originated. These findings support the potential therapeutic relevance of developing strategies to undermine the progression of normal cells to mesenchymal transition without subverting cell viability.

Lenticular mitoprotection. Part B: GSK-3ß and regulation of mitochondrial permeability transition for lens epithelial cells in atmospheric oxygen.

PURPOSE: Loss of integrity of either the inner or outer mitochondrial membrane results in the dissipation of the mitochondrial electrochemical gradient that leads to mitochondrial membrane permeability transition (mMPT). This study emphasizes the role of glycogen synthase kinase 3beta (GSK-3ß) in maintaining mitochondrial membrane potential, thus preventing mitochondrial depolarization (hereafter termed mitoprotection). Using 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione (SB216763), an inhibitor of GSK-3ß, and drawing a distinction between it and 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (UO126), an inhibitor of extracellular-signal-regulated kinase (ERK) phosphorylation, the means by which GSK-3ß influences mitoprotection in cultured human lens epithelial (HLE-B3) cells and normal, secondary cultures of bovine lens epithelial cells, maintained in atmospheric oxygen, was investigated. METHODS: Virally transfected human lens epithelial cells (HLE-B3) and normal cultures of bovine lens epithelial cells were exposed to acute hypoxic conditions (about 1% O2) followed by exposure to atmospheric oxygen (about 21% O2). Specific antisera and western blot analysis was used to examine the state of phosphorylation of ERK and GSK-3ß, as well as the phosphorylation of a downstream substrate of GSK-3ß, glycogen synthase (GS, useful in monitoring GSK-3ß activity). The potentiometric dye, 1H-benzimidazolium-5,6-dichloro-2-[3-(5,6-dichloro-1,3-diethyl-1,3-dihydro-2H-benzimidazol-2-ylidene)-1-propenyl]-1,3-diethyl-iodide (JC-1), was used to monitor mitochondrial depolarization upon exposure of inhibitor treatment relative to the control cells (mock inhibition) in atmospheric oxygen. Caspase-3 activation was scrutinized to determine whether mitochondrial depolarization inevitably leads to apoptosis. RESULTS: Treatment of HLE-B3 cells with SB216763 (12 µM) inactivated GSK-3ß activity as verified by the enzyme's inability to phosphorylate its substrate, GS. SB216763-treated cells were not depolarized relative to the control cells as demonstrated with JC-1 fluorescent dye analysis. The HLE-B3 cells treated with UO126, which similarly blocked phosphorylation of GS, were nevertheless prone to mMPT relative to the control cells. Western blot analysis determined that Bcl-2-associated X (BAX) levels were unchanged for SB216763-treated or UO126-treated HLE-B3 cells when compared to their respective control cells. However, unlike the SB216763-treated cells, the UO126-treated cells showed a marked absence of Bcl-2, as well as phosphorylated Bcl-2 relative to the controls. UO126 treatment of bovine lens epithelial cells showed similar results with pBcl-2 levels, while the Bcl-2 content appeared unchanged relative to the control cells. HLE-B3 and normal bovine lens cell cultures showed susceptibility to mMPT associated with the loss of pBcl-2 by UO126 treatment. CONCLUSIONS: MITOCHONDRIAL DEPOLARIZATION MAY OCCUR BY ONE OF TWO KEY OCCURRENCES: interruption of the electrochemical gradient across the inner mitochondrial membrane resulting in mMPT or by disruption of the integrity of the inner or outer mitochondrial membrane. The latter scenario is generally tightly regulated by members of the Bcl-2 family of proteins. Inhibition of GSK-3ß activity by SB216763 blocks mMPT by preventing the opening of the mitochondrial permeability transition pore. UO126, likewise, inhibits GSK-3ß activity, but unlike SB216763, inhibition of ERK phosphorylation induces the loss of intracellular pBcl-2 levels under conditions where intracellular BAX levels remain constant. These results suggest that the lenticular mitoprotection normally afforded by the inactivation of GSK-3ß activity may, however, be bypassed by a loss of pBcl-2, an anti-apoptotic member of the Bcl-2 family. Bcl-2 prevents the translocation of BAX to the mitochondrial outer membrane inhibiting depolarization by disrupting the normal electrochemical gradient leading to mMPT.

Lenticular mitoprotection. Part A: Monitoring mitochondrial depolarization with JC-1 and artifactual fluorescence by the glycogen synthase kinase-3ß inhibitor, SB216763.

PURPOSE: Dissipation of the electrochemical gradient across the inner mitochondrial membrane results in mitochondrial membrane permeability transition (mMPT), a potential early marker for the onset of apoptosis. In this study, we demonstrate a role for glycogen synthase kinase-3ß (GSK-3ß) in regulating mMPT. Using direct inhibition of GSK-3ß with the GSK-3ß inhibitor SB216763, mitochondria may be prevented from depolarizing (hereafter referred to as mitoprotection). Cells treated with SB216763 showed an artifact of fluorescence similar to the green emission spectrum of the JC-1 dye. We demonstrate the novel use of spectral deconvolution to negate the interfering contributing fluorescence by SB216763, thus allowing an unfettered analysis of the JC-1 dye to determine the mitochondrial membrane potential. METHODS: Secondary cultures of virally transfected human lens epithelial cells (HLE-B3) were exposed to acute hypoxic conditions (approximately 1% O2) followed by exposure to atmospheric oxygen (approximately 21% O2). The fluorescent dye JC-1 was used to monitor the extent of mitochondrial depolarization upon exposure of inhibitor treatment relative to the control cells (mock inhibition) in atmospheric oxygen. Annexin V-fluorescein isothiocyanate/propidium iodide staining was implemented to determine cell viability. RESULTS: Treatment of HLE-B3 cells with SB216763 (12 µM), when challenged by oxidative stress, suppressed mitochondrial depolarization relative to control cells as demonstrated with JC-1 fluorescent dye analysis. Neither the control nor the SB216763-treated HLE-B3 cells tested positive with annexin V-fluorescein isothiocyanate/propidium iodide staining under the conditions of the experiment. CONCLUSIONS: Inhibition of GSK-3ß activity by SB216763 blocked mMPT relative to the slow but consistent depolarization observed with the control cells. We conclude that inhibition of GSK-3ß activity by the GSK-3ß inhibitor SB216763 provides positive protection against mitochondrial depolarization.

Lenticular cytoprotection. Part 1: the role of hypoxia inducible factors-1α and -2α and vascular endothelial growth factor in lens epithelial cell survival in hypoxia.

PURPOSE: The prosurvival signaling cascades that mediate the unique ability of human lens epithelial cells to survive in their naturally hypoxic environment are not well defined. Hypoxia induces the synthesis of the hypoxia inducible factor HIF-1α that in turn, plays a crucial role in modulating a downstream survival scheme, where vascular endothelial growth factor (VEGF) also plays a major role. To date, no published reports in the lens literature attest to the expression and functionality of HIF-2α and the role it might play in regulating VEGF expression. The aim of this study was to identify the functional expression of the hypoxia inducible factors HIF-1α and HIF-2α and establish their role in regulating VEGF expression. Furthermore, we demonstrate a link between sustained VEGF expression and the ability of the hypoxic human lens epithelial cell to thrive in low oxygen conditions and resist mitochondrial membrane permeability transition (also referred to as lenticular cytoprotection). METHODS: Hypoxia inducible factor translation inhibitors were used to demonstrate the role of HIF-1α and HIF-2α and the simultaneous expression of both hypoxic inducible factors to determine their role in regulating VEGF expression. Axitinib, which inhibits lenticular cell autophosphorylation of its VEGF receptor, was employed to demonstrate a role for the VEGF-VEGFR2 receptor complex in regulating Bcl-2 expression. Specific antisera and western blot analysis were used to detect the protein levels of HIF-1α and HIF-2α, as well as the proapoptotic protein, BAX and the prosurvival protein, Bcl-2. VEGF levels were analyzed with enzyme-linked immunosorbent assay (ELISA). The potentiometric dye, 5,5',6,6'-tetrachloro1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine iodide, was used to determine the effect of the inhibitors on mitochondrial membrane permeability transition. RESULTS: Cultured human lens epithelial cells (HLE-B3) maintained under hypoxic condition (1% oxygen) displayed consistent accumulation of VEGF throughout the 72 h incubation period. Using hypoxia inducible factor translation inhibitors targeting HIF-1α or HIF-2α, the specific inhibition of each protein did not diminish VEGF synthesis. The combined inhibition of HIF-1α and HIF-2α expression, using a double hypoxia inducible factor translation inhibitor, markedly decreased the level of VEGF. The inhibition of VEGF synthesis was associated with a profound deficiency in the level of the prosurvival protein, Bcl-2. Axitinib also prevented the VEGF-mediated expression of Bcl-2. The loss of VEGF coupled with the decrease in intracellular Bcl-2 correlated with marked mitochondrial depolarization, an early predictor of cellular apoptosis. CONCLUSIONS: Our data support a model in which the sustained synthesis of VEGF in human lens epithelial cells, maintained under hypoxic condition, is regulated by a compensatory inter-relationship between HIF-1α and HIF-2α. VEGF acts as a prosurvival factor in hypoxic lens epithelial cells by maintaining consistent expression of the prosurvival protein Bcl-2, which likely prevents the translocation of cytosolic BAX to the outer mitochondrial membrane, thus preventing the initiation of mitochondrial depolarization.

Functional 20S proteasomes in mature human red blood cells.

The purpose of the present study was to investigate whether functional 20S and/or 26S proteasomes are present within mature human red blood cells (RBCs; depleted of reticulocytes and leukocytes). Double-immunofluorescence confocal microscopy showed the presence of immunoreactive 20S and 19S proteasomal subunit proteins and their partial co-localization within mature RBCs. Proteasomes isolated from mature RBCs displayed 20S activity in vitro; atomic-force and transmission electron microscopy of isolated proteasomes revealed abundant 20S core particles and very few 26S particles. A two-dimensional differential in-gel electrophoresis (2D-DIGE) approach was used to determine if proteasome-dependent protein degradation occurs within mature RBCs. Twenty-eight proteins were identified with altered protein content in response to lactacystin. Seven cytosolic proteins showed an increase and 16 showed a decrease; five membrane proteins showed a decrease. We conclude that the proteins showing increased abundance are either primary or secondary targets of the 20S proteasome and that putatively degraded proteins are secondary targets. Therefore, functional 20S proteasomes exist within mature RBCs. Our study did not detect 26S proteasome activity using the 2D-DIGE approach.

Amoebicidal activities of alexidine against 3 pathogenic strains of acanthamoeba.

OBJECTIVES: Effective pharmacotherapy for Acanthamoeba keratitis has been hampered because of the marked resistance of various stains to a variety of antimicrobial agents. In view of the fact that topical Brolene (propamidine isethionate) and neosporin are currently considered to be the first-line medical treatment of choice in Europe, we sought to determine whether Alexidine is equally effective, because the latter drug is more readily available in the United States. METHODS: Trophozoites and cysts from 3 pathogenic corneal isolates (A. castellanii, A. polyphaga, and A. rhysodes) were incubated in peptone-yeast extract-glucose medium containing different concentrations of Alexidine for 24 hr. The number of trophozoites was counted by hemocytometer. The cysts were plated in to nonnutrient agar plates precoated with Escherichia coli and observed for viability or excystment over a period of 2 weeks. The capacity of different concentrations of Alexidine to induce cytolysis of corneal epithelial cells was tested in vitro. Chinese hamster corneas were treated with 5 microL of Alexidine topically, every hour; 6 times a day and the corneas were stained with fluorescein to asses the epithelial defects in vivo. RESULTS: Alexidine was effective in killing the trophozoites at a concentration of 10 microg/mL. However, a higher concentration of Alexidine (100 microg/mL) is required to kill Acanthamoeba cysts and the cytotoxic activities of Alexidine are comparable with chlorhexidine. We have also demonstrated that both Alexidine and chlorhexidine at 100 microg/mL induced significant cytopathic effect on the corneal epithelial cells in vitro. In vivo results indicate that Alexidine at a concentration of 100 microg/mL is less toxic than chlorhexidine when applied topically to the Chinese hamster cornea. CONCLUSIONS: Our study has identified Alexidine as a novel anti-Acanthamoeba drug and suggests that Alexidine may be an effective therapeutic option because of its potency and low toxicity to the corneal tissues when applied topically in vivo.

Modulation of corneal and stromal matrix metalloproteinase by the mannose-induced Acanthamoeba cytolytic protein.

The involvement of the mannose-induced Acanthamoeba cytopathic protein (MIP-133) in tissue injury and activation of metalloproteinase of corneal and stromal cells was examined in vitro. Activation of MMP-1, MMP-2, MMP-3, and MMP-9 induced by MIP-133 on human corneal epithelial and stromal cell cultures was examined by reverse transcriptase polymerase chain reaction (RT-PCR) and ELISA. MMP-1, MMP-2, MMP-3, and MMP-9 mRNA were expressed in both cultured human corneal epithelial and stromal cells. When the epithelial cells were exposed to MIP-133 protein, the mRNA expression for MMP-1 and MMP-9 was unchanged. However, the transcript for MMP-2 and MMP-3 was decreased by 2-fold. By contrast, the expression of MMP-2 and MMP-3 was significantly upregulated (2- to 4-fold) in the corneal stromal cells 1, 4, and 8h after MIP-133 stimulation. At the protein level, there was no significant difference in the level of MMPs between the corneal epithelial cells before and after stimulation with MIP-133. By contrast, the levels of MMP-2 and MMP-3 were significantly higher in the corneal stromal cells stimulated with MIP-133. The supernatants from corneal stromal cells stimulated with MIP-133 were incubated with PMSF and MIP-133 antibody and the level of MMP-2 was measured by ELISA. Activation of MMP-2 by MIP-133 was inhibited in the supernatants pretreated with the serine protease inhibitor, PMSF, and anti-MIP-133. Supernatants pretreated with the cysteine protease inhibitor E6 or control antibody produced the same amount of MMP-2 as the untreated supernatants. To verify possible homology between MMPs and Acanthamoeba castellanii proteases, the mRNA from A. castellanii was prepared and analyzed for the expression of MMP genes by PT-PCR. The results showed that A. castellanii did not express mRNA for MMP-1, MMP-2, MMP-3, or MMP-9. Thus, A. castellanii mRNA does not cross-react with human MMPs. Furthermore, ELISA was used to determine the cross-reactivity of MMP antibodies with the MIP-133 protein. Monoclonal antibodies against MMPs did not cross-react with either the MIP-133 protein or BSA (negative control antigen). The results indicate that the MIP-133 protein modulates MMP-2 and -3 expression differently in human corneal epithelial and stromal cells.

Effect of immunization with the mannose-induced Acanthamoeba protein and Acanthamoeba plasminogen activator in mitigating Acanthamoeba keratitis.

PURPOSE: The mannose-induced cytopathic protein (MIP-133) and Acanthamoeba plasminogen activator (aPA) play key roles in the pathogenesis of Acanthamoeba keratitis by inducing a cytopathic effect on the corneal epithelial and stromal cells and by production of proteolytic enzymes that facilitate the invasion of trophozoites through the basement membrane. The goal of the present study was to gain insight into the pathogenicity of Acanthamoeba infection as well as to determine whether oral immunization with aPA and MIP-133 produce an additive protection against Acanthamoeba keratitis. METHODS: MIP-133 and aPA were isolated by chromatography. The purity of the concentrated MIP-133 and aPA was confirmed by SDS-PAGE and fibrinolytic activity, respectively. aPA activity of Acanthamoeba cultures was quantitated by radial diffusion in fibrin-agarose gel. The capacity of aPA and MIP-133 to induce cytolysis of corneal epithelial cells was tested in vitro. Chinese hamsters were orally immunized with four weekly doses of aPA or MIP-133 conjugated with cholera toxin. The animals were immunized before infection to determine the prophylactic effect of oral immunization. The therapeutic effect of oral immunization with aPA and MIP-133 was determined after corneal infection had been established. The animals were then infected via Acanthamoeba castellanii-laden contact lenses. RESULTS: aPA was characterized in pathogenic and nonpathogenic strains of Acanthamoeba spp. Oral immunization with MIP-133 before and after infection with Acanthamoeba significantly reduced the severity of corneal infection which includes infiltration and ulceration (P < 0.05) and shortened the duration of the disease. Immunization with aPA alone did not significantly affect the course of disease (P > 0.05). CONCLUSIONS: These data suggest that once trophozoites invade the cornea, MIP-133 production is necessary to initiate corneal disease and plays an important role in the subsequent steps of the pathogenic cascade of Acanthamoeba keratitis.

The isolation of reticulocyte-free human red blood cells.

We depleted reticulocytes from erythrocytes of both sickle cell disease (SCD) subjects and healthy controls by four methods: fluorescence-activated cell sorting (FACS), Miltenyi immunomagnetic depletion (MACS), a combination of these methods (FACS + MACS) and Percoll density separation. The efficiency of these methods was assessed by new methylene blue staining and manual enumeration of the reticulocytes. FACS sorted erythrocytes from reticulocytes based on size and granularity, as well as the absence of dsDNA staining. MACS depleted reticulocytes from erythrocytes based on the immunoaffinity to CD36 and CD71. Reticulocytes from healthy controls were depleted to

Role of activated macrophages in Acanthamoeba keratitis.

The purpose of this study was to determine whether activating the conjunctival macrophages would affect the course of Acanthamoeba spp. keratitis in a Chinese hamster model of this disease. Chinese hamster spleen cells were stimulated with concanavalin A (Con A), and interferon gamma (IFN-gamma) -containing supernatants were collected 24 hr later. The IFN-gamma-containing supernatants were loaded into liposomes, which were fed to peritoneal macrophages in vitro. Macrophage activation was assessed by testing for production of nitric oxide (NO) with the use of Griess reagent. Conjunctival macrophages were activated in situ by subconjunctival injection of liposomes containing Con A-activated spleen cell culture supernatants. Control liposomes were loaded with phosphate-buffered saline (PBS). Macrophages exposed to supernatants from Con A-stimulated spleen cells produced 4-fold-higher amounts of NO than unstimulated macrophages. Activation of macrophages via subconjunctival injection of liposomes containing supernatants from Con A-stimulated spleen cell cultures resulted in rapid resolution of the corneal infection. Approximately 80% of animals treated with PBS-containing liposomes demonstrated evidence of corneal disease at day 14 compared to 10% incidence of infection in the Con A-treated group. Moreover, at all time points examined, the clinical appearance of the keratitis in animals treated with liposomes containing Con A supernatant was significantly reduced compared to the group treated with liposomes containing PBS (P < 0.05). Macrophages stimulated with IFN-gamma-containing supernatants killed significant numbers of the trophozoites in vitro (P < 0.05). Killing was inhibited by cytochalasin D, but not by L-N6-1-iminoethyl-L-lysine dihydrochloride (L-NIL), which is a selective inhibitor of inducible NO synthase (INOS).

Downregulation of survivin expression enhances sensitivity of cultured uveal melanoma cells to cisplatin treatment.

The purpose of this study is to evaluate the effect of a novel anti-apoptotic gene, survivin, on the resistance and susceptibility of human uveal melanoma cells to apoptosis induced by cisplatin. The sensitivity of human uveal melanoma cell lines to apoptosis induced by cisplatin was analyzed by caspase-3 assays. The expression of the anti-apoptotic protein, survivin, was examined by flow cytometry. Melanoma cells were transfected with either survivin cDNA or survivin anti-sense cDNA and examined for susceptibility to cisplatin-induced apoptosis. Six human uveal melanoma cell lines were incubated with or without cisplatin and cellular proliferation was determined by MTT assays. Significant growth inhibition was observed in 3 melanoma cell lines (OMM1, OCM3, and MEL 270). By contrast, 3 cell lines (OMM2.5, OMM2.3, and 92-1) were resistant to cisplatin-induced apoptosis. However, a positive association was observed between resistance to cisplatin-induced apoptosis and high expression of the anti-apoptotic protein, survivin. Up-regulation of survivin by gene transfer enhanced resistance to cisplatin-induced apoptosis, while transfection with survivin anti-sense rendered resistant melanoma cells susceptible to cisplatin. The combination of cisplatin and actinomycin D significantly decreased survivin expression and enhanced the cisplatin-induced apoptosis of uveal melanoma cells in vitro. These data indicate that resistance of some uveal melanoma cells to cisplatin-induced apoptosis is controlled by anti-apoptotic proteins, such as survivin, that are sensitive to actinomycin D treatment.