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PurposeThe integrin αvβ3 is differentially expressed on neovascular endothelial cells. We investigated whether a novel intravenously injectable αvβ3 integrin-ligand coupled nanoparticle (NP) can target choroidal neovascular membranes (CNV) for imaging and targeted gene therapy.MethodsCNV lesions were induced in rats using laser photocoagulation. The utility of NP for in vivo imaging and gene delivery was evaluated by coupling the NP with a green fluorescing protein plasmid (NP-GFPg). Rhodamine labeling (Rd-NP) was used to localize NP in choroidal flatmounts. Rd-NP-GFPg particles were injected intravenously on weeks 1, 2, or 3. In the treatment arm, rats received NP containing a dominant negative Raf mutant gene (NP-ATPμ-Raf) on days 1, 3, and 5. The change in CNV size and leakage, and TUNEL positive cells were quantified.ResultsGFP plasmid expression was seen in vivo up to 3 days after injection of Rd-NP-GFPg. Choroidal flatmounts confirmed the localization of the NP and the expression of GFP plasmid in the CNV. Treating the CNV with NP-ATPμ-Raf decreased the CNV size by 42% (PConclusionSystemic administration of αvβ3 targeted NP can be used to label the abnormal blood vessels of CNV for imaging. Targeted gene delivery with NP-ATPμ-Raf leads to a reduction in size and leakage of the CNV by induction of apoptosis in the CNV.

Matrix metalloproteinase-9 (MMP-9) plays a critical role in tissue remodeling under both physiological and pathological conditions. Although MMP-9 expression is low in most cells and is tightly controlled, the mechanism of its regulation is poorly understood. We utilized mouse embryonic fibroblasts (MEFs) that were nullizygous for the catalytic α subunit of AMP-activated protein kinase (AMPK), which is a key regulator of energy homeostasis, to identify AMPK as a suppressor of MMP-9 expression. Total AMPKα deletion significantly elevated MMP-9 expression compared with wild-type (WT) MEFs, whereas single knock-out of the isoforms AMPKα1 and AMPKα2 caused minimal change in the level of MMP-9 expression. The suppressive role of AMPK on MMP-9 expression was mediated through both its activity and presence. The AMPK activators 5-amino-4-imidazole carboxamide riboside and A769662 suppressed MMP-9 expression in WT MEFs, and AMPK inhibition by the overexpression of dominant negative (DN) AMPKα elevated MMP-9 expression. However, in AMPKα(-/-) MEFs transduced with DN AMPKα, MMP-9 expression was suppressed. AMPKα(-/-) MEFs showed increased phosphorylation of IκBα, expression of IκBα mRNA, nuclear localization of nuclear factor-κB (NF-κB), and DNA-binding activity of NF-κB compared with WT. Consistently, selective NF-κB inhibitors BMS345541 and SM7368 decreased MMP-9 expression in AMPKα(-/-) MEFs. Overall, our results suggest that both AMPKα isoforms suppress MMP-9 expression and that both the activity and presence of AMPKα contribute to its function as a regulator of MMP-9 expression by inhibiting the NF-κB pathway.

Background Detachment of photoreceptors from the underlying retinal pigment epithelium is seen in various retinal disorders such as retinal detachment and age-related macular degeneration and leads to loss of photoreceptors and vision. Pharmacologic inhibition of photoreceptor cell death may prevent this outcome. This study tests whether systemic administration of tauroursodeoxycholic acid (TUDCA) can protect photoreceptors from cell death after experimental retinal detachment in rodents. Methodology/principal findings Retinal detachment was created in rats by subretinal injection of hyaluronic acid. The animals were treated daily with vehicle or TUDCA (500 mg/kg). TUNEL staining was used to evaluate cell death. Photoreceptor loss was evaluated by measuring the relative thickness of the outer nuclear layer (ONL). Macrophage recruitment, oxidative stress, cytokine levels, and caspase levels were also quantified. Three days after detachment, TUDCA decreased the number of TUNEL-positive cells compared to vehicle (651±68/mm(2) vs. 1314±68/mm(2), P = 0.001) and prevented the reduction of ONL thickness ratio (0.84±0.03 vs. 0.65±0.03, P = 0.002). Similar results were obtained after 5 days of retinal detachment. Macrophage recruitment and expression levels of TNF-a and MCP-1 after retinal detachment were not affected by TUDCA treatment, whereas increases in activity of caspases 3 and 9 as well as carbonyl-protein adducts were almost completely inhibited by TUDCA treatment. Conclusions/significance Systemic administration of TUDCA preserved photoreceptors after retinal detachment, and was associated with decreased oxidative stress and caspase activity. TUDCA may be used as a novel therapeutic agent for preventing vision loss in diseases that are characterized by photoreceptor detachment.

PURPOSE To describe the in vivo evolution of laser-induced choroidal neovascularization (CNV) in mice using spectral domain optical coherence tomography (SD-OCT). METHODS Laser photocoagulation was applied to the mouse fundus using a 532-nm diode laser (100, 150, and 200 mW; 100-μm diameter, 0.1-second duration). SD-OCT examination was performed immediately after laser application and at days 3, 5, 7, 14, 21, and 28 after laser. Fluorescein angiography (FA) was performed at day 5, 7, 14, and 28. Acquired SD-OCT images were analyzed to describe morphologic features, measure CNV size and retinal thickness, and assess the frequency of lesions resulting in fluid accumulation. Finally, SD-OCT images were compared to fluorescein angiograms and histologic sections with immunostaining at similar time points. RESULTS SD-OCT allowed visualization of the initial laser damage and the subsequent stages of the injury response. CNV formation reached its maximum size at day 5. By day 7, significant size reduction was observed (P < 0.001), continuing through days 14 and 28. Exudation signs, such as fluid accumulation and increase in retinal thickness, followed the same time course, with a peak at day 5 and a decrease by day 7. Delivery of higher laser energy levels to the RPE/choroid complex resulted in a significant percentage of lesions demonstrating excessive chorioretinal damage without CNV formation. CONCLUSIONS SD-OCT is a fast and reliable tool for the in vivo evaluation of laser-induced CNV, allowing quantification of lesion size and exudation parameters. Moreover, it provides morphologic information that correlates with histologic findings.

Purpose: The purpose was to study the clinical profile and prognosis of young patients with uveal melanoma treated by proton beam irradiation. Methods: A retrospective case-control series was studied. Seventeen patients aged ≤20 years with uveal melanoma and 51 risk factor-matched control subjects aged >20 years were treated for posterior uveal melanoma between 1975 and 2004. All patients were treated with proton beam irradiation at Harvard Cyclotron. Metastasis and survival of patients were the main outcome measures. Results: Of a total of 3,207 patients with uveal melanoma, 17 patients were ≤20 years of age at the time of diagnosis. No metastatic deaths were observed (median follow-up, 16 years). Among the control group, there were 7 of 50 metastasis-related deaths. Conclusion: There was an excellent outcome regarding metastasis in a cohort of young patients with posterior uveal melanoma treated with proton beam irradiation.

PURPOSE To identify a novel, sensitive, nonradioactive leakage assay that can be used in the assessment of retinal vascular permeability in rats and mice. METHODS Breakdown of the vascular barrier was induced by vascular endothelial growth factor (VEGF), lipopolysaccharide (LPS), or diabetes. Biotinylated bovine serum albumin (bBSA) was administered as a tracer. After perfusion with lactated Ringer's solution, extravasated bBSA was detected with immunoprecipitation and Western blot analysis or sandwich ELISA. The results were then normalized against the final bBSA plasma concentration, the circulation time, and the protein concentration of the tissue. RESULTS Six hours after VEGF injection, BRB breakdown was quantified in the injected eye and was 2.5-fold higher than in the contralateral phosphate-buffered saline (PBS)-injected eye (n = 6 rats, P < 0.01). Intravitreal LPS injection induced severe inflammation in the directly injected eye and moderate inflammation in the contralateral untreated eye. Leakage was six- and threefold higher, respectively, compared with that in the untreated control animals (n = 5 rats, P < 0.01). Nine-month diabetic rats had a threefold increase in vascular leakage compared with age-matched control animals (n = 6 retinas, P < 0.05). Twenty-four hours after intraperitoneal administration of LPS in mice, the animals showed increased vascular leakage in all tissue organs examined (retina, 1.7-fold; brain, 1.5-fold; and kidney, 1.3-fold). CONCLUSIONS bBSA can serve as an effective alternative to the current methods used for quantitating vascular leakage and especially the blood-retinal barrier breakdown. It is reasonably easy to perform, low in cost, and adaptable to experiments in mice.

Macroaneurysms are saccular dilatations of the retinal arterioles commonly seen in systemic hypertension. The authors present the first report of the high-resolution optical coherence tomography appearance of an unruptured retinal artery macroaneurysm.

Purpose To determine the percentage of patients in whom endophthalmitis developed after open globe injury. Design Retrospective, noncomparative, consecutive case series. Methods Charts of all patients (675 in total) treated surgically for open globe injury at the Massachusetts Eye and Ear Infirmary (MEEI) between January 1, 2000 and July 31, 2007 were reviewed. Cases with at least 30 days of follow-up were included in statistical analyses (558 in total). A standardized treatment protocol was used in all cases. Intravenous vancomycin and ceftazidime were started on admission and were stopped after 48 hours. Patients were discharged on topical antibiotics, corticosteroids, and cycloplegia. Surgical repairs were performed by the chief of trauma, a full-time position rotating yearly, who is on call for all open globe trauma. Data collection variables included timing of injury and repair, mechanism of injury, details of surgical repair, and details of follow-up such as duration, presence of complications, and vision. A primary outcome measure of endophthalmitis and secondary outcome measure of risk factors for endophthalmitis were studied. Results During 7.5 years, 675 open globe injuries were treated at MEEI. Of these, 558 had at least 30 days of follow-up (mean, 11 months) and were used in statistical analyses. The overall percentage of endophthalmitis was 0.9% (3 culture-positive cases and 2 culture-negative cases). Four of the 5 cases achieved final acuity of 20/80 or better. Risk factors for endophthalmitis included intraocular foreign body ( P = .03; odds ratio, 7.52) and primary intraocular lens placement ( P = .05). Conclusions A standardized protocol including surgical repair by a dedicated eye trauma service and 48 hours of intravenous antibiotics was associated with a posttraumatic endophthalmitis percentage of less than 1%.

In recent years, research of degenerative retinal diseases has been focused on understanding cell death and ways of interfering with it. Several experimental animal models of diabetic retinopathy, retinitis pigmentosa, retinal detachment, and glaucoma, have revealed that cell death in the retina can occur through different modalities, including apoptosis and necroptosis among others, and encompasses multiple inducers and pathways and displays different morphologic characteristics.1–5 From the many tools used to characterize and quantify cell death, the terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) assay is likely the most widespread method. This assay detects fragmented or nicked DNA by means of a terminal deoxynucleotidyl transferase enzyme, which incorporates fluorescent-labeled dUTPs in damaged nucleic acid regions. First described by Gavrieli et al.,6 this assay has been a sensitive method for detecting apoptosis.7 However, as TUNEL assay detects DNA fragments regardless of the induced cell death pathway, it cannot distinguish between apoptotic or other forms of programmed cell death.3,8–10 Despite its limitations, TUNEL assay remains the most widespread method used to screen for any form of programmed cell death, to date. In order to quantitate TUNEL assay results in tissue sections, manual counting of TUNEL-positive (TUNEL+) cells, ideally by two masked observers is performed.11 Observers count TUNEL+ cells and measure the respective retinal area, and results are expressed as either TUNEL+ cells/area or TUNEL+ cells/total cells. However, this process is time consuming, prone to measurement errors, and not entirely reproducible. Therefore, an automated approach is of interest to address these difficulties. To the best of our knowledge, there is no automated cell death quantitation method available, capable of segmenting retinal layers and counting individual TUNEL+ cells in the retina without performance variability. The purpose of this study was to introduce a novel ImageJ macro for the automated detection of cell death in retinal cross-sections. In addition, we studied differences in grading between an experienced and an inexperienced observer. Because the introduction of a new measurement method requires that it be parallel with the gold standard, or commonly accepted method, we validated this macro by comparing its performance relative to that of two observers. We found that the ImageJ macro can achieve a fast and accurate retinal cell death quantitation.

A, A 33-year-old woman presented with decreased vision in the right eye, pain, and a white subretinal elevated lesion. Fluorescein angiography showed diffuse late leakage (B) and optical coherence tomography revealed elevated choroid and subretinal fluid (C). D, An orbital magnetic resonance image displayed a hyperintense signal (arrowhead). JAMA Ophthalmol. 2015;133(1):e141801. doi:10.1001/jamaophthalmol.2014.1801

Purpose To report 2 patients with combined intraocular and orbital vascular abnormalities. Design Two interventional case reports. Main outcome measures Clinical and pathologic findings. Intervention/testing Orbitotomy, fluorescein angiography, magnetic resonance imaging, and radiation therapy. Results Two patients presented with vision loss, intermittent proptosis, and cosmetic deformity. These patients had orbital lymphangioma, ipsilateral retinal and iris vascular malformations, and smaller corneal diameter on the affected side. Conclusions The coexistence of these diverse vascular anomalies supports the established hypothesis of pluripotential orbital vascular anlagen. Disruptive influences of various types during embryogenesis or development may produce a variety of congenital orbital and intraocular vascular malformations.

To evaluate the potential for mouse genetic background to effect photoreceptor cell death in response to experimental retinal detachment (RD).Retinal detachment was induced in three inbred mouse strains (C57BL/6, BALB/c, and B6129SF2) by subretinal injection of sodium hyaluronate. A time course of photoreceptor cell death was assessed by TUNEL assay. Total photoreceptor cell death was analyzed through comparing the outer nuclear layer (ONL)/inner nuclear layer (INL) ratio 7 days post RD. Western blot analysis or quantitative real-time PCR (qPCR) were performed to assess cell death signaling, expression of endogenous neurotrophin, and levels of apoptosis inhibitors 24 hours after RD. Inflammatory cytokine secretion and inflammatory cell infiltration were quantified by ELISA and immunostaining, respectively.The peak of photoreceptor cell death after RD was at 24 hours in all strains. Photoreceptor cell death as well as monocyte chemoattractant protein 1 and interleukin 6 secretion at 24 hours after RD was the highest in BALB/c, followed in order of magnitude by C57BL/6 and B6129SF2. Conversely, nerve growth factor expression and ONL/INL ratio were the lowest in BALB/c. Apoptosis signaling was higher in C57BL/6, whereas necroptosis signaling was higher in C57BL/6 and BALB/c. Autophagic signaling was higher in BALB/c. X-linked inhibitor of apoptosis (XIAP) and survivin protein levels were lower in C57BL/6 and BALB/c, respectively. Macrophage/microglia infiltration was higher in C57BL/6 and BALB/c at 24 hours after RD.Photoreceptor cell death after RD was significantly different among the three strains, suggesting the presence of genetic factors that affect photoreceptor cell death after RD.

Purpose To evaluate the effects and mechanism of aminoimidazole carboxamide ribonucleotide (AICAR), an AMP-dependent kinase (AMPK) activator, on the growth of uveal melanoma cell lines. Methods Four different cell lines were treated with AICAR (1-4 mM). Cell growth was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. Cell cycle analysis was conducted by flow cytometry; additionally, expression of cell-cycle control proteins, cell growth transcription factors, and downstream effectors of AMPK were determined by RT-PCR and Western blot. Results Aminoimidazole carboxamide ribonucleotide inhibited cell growth, induced S-phase arrest, and led to AMPK activation. Aminoimidazole carboxamide ribonucleotide treatment was associated with inhibition of eukaryotic translation initiation factor 4E-BP1 phosphorylation, a marker of mammalian target of rapamycin (mTOR) pathway activity. Aminoimidazole carboxamide ribonucleotide treatment was also associated with downregulation of cyclins A and D, but had minimal effects on the phosphorylation of ribosomal protein S6 or levels of the macroautophagy marker LC3B. The effects of AICAR were abolished by treatment with dipyridamole, an adenosine transporter inhibitor that blocks the entry of AICAR into cells. Treatment with adenosine kinase inhibitor 5-iodotubericidin, which inhibits the conversion of AICAR to its 5'-phosphorylated ribotide 5-aminoimidazole-4-carboxamide-1-D-ribofuranosyl-5'-monophosphate (ZMP; the direct activator of AMPK), reversed most of the growth-inhibitory effects, indicating that some of AICAR's antiproliferative effects are mediated at least partially through AMPK activation. Conclusions Aminoimidazole carboxamide ribonucleotide inhibited uveal melanoma cell proliferation partially through activation of the AMPK pathway and downregulation of cyclins A1 and D1.

We read with interest the article by Reznicek et al, 1 which evaluated the fulfilment of retreatment criteria for recurrent neovascular age-related macular degeneration (AMD). We support the notion of qualitative optical coherence tomography (OCT) assessment of the retina. However, we will like to point out some factors that might have affected their conclusion that quantitative OCT-based retreatment criteria appeared not sensitive enough in a clinical setting. Reznicek et al 1 mentioned that Stratus-OCT was used in the …

We read with interest on the article by Karabas et al 1, which reported statistically significant decreased subfoveal choroidal thickness in patients with oculocutaneous albinism (OCA) as compared with age-controlled normal subjects. We would like to congratulate the authors on being the first to describe this among OCA subjects. However, there are relevant factors that we will like to point out. Karabas pointed out that their findings of choroidal thickness among the healthy control population were higher than other studies. Karabas reported …

EGFL7 is a secreted angiogenic factor, which in contrast to the well-known secreted angiogenic molecules VEGF and FGF-2, is almost exclusively expressed by endothelial cells and may act in an autocrine fashion. Prior studies have shown EGFL7 to mediate its angiogenic effects by interfering with the Notch pathway and/or via the intronic miR126. Less is known about its effects on VEGF signaling. We wanted to investigate the role of epidermal growth factor-like domain 7 (EGFL7) in VEGF-driven angiogenesis using an ex vivo Matrigel-embedded mouse eye cup assay and siRNA mediated knockdown of EGFL7 by siRNA. Our results suggested that VEGF-induced vascular tube formation was significantly impaired after siRNA downregulation of EGFL7. In addition, knockdown of EGFL7 suppressed VEGF upregulation of phospho-Akt and phospho-Erk(1/2) in endothelial cells, but did not alter VEGFR phosphorylation and neuropilin-1 protein expression or miR126 expression. Thus, in conclusion, EGFL7 is required for VEGF upregulation of the Akt/Erk (1/2) pathway during angiogenesis, and may represent a new therapeutic target in diseases of pathological neovascularization.

Malonyl CoA is a regulator of carnitine palmitoyl transferase 1 (CPT1), the enzyme that controls the transfer of long chain fatty acyl CoA into mitochondria where it is oxidized. Recent studies indicate that in skeletal muscle the concentration of malonyl CoA is acutely (minutes) regulated by changes in its fuel supply and energy expenditure. In response to changes in fuel supply, regulation appears to be due to alterations in the cytosolic concentration of citrate, which is both an allosteric activator of acetyl CoA carboxylase (ACC), the enzyme that catalyzes malonyl CoA synthesis and a source of its precursor, cytosolic acetyl CoA. During exercise and immediately thereafter regulation by citrate appears to be lost and malonyl CoA levels diminish as the result of a decrease in ACC activity secondary to phosphorylation. Sustained increases in the concentration of malonyl CoA have been observed in muscle of a number of insulin-resistant rodents including the Zucker (fa/fa) and GK rats, KKAy mice, glucose-infused rats and rats in which muscle has been made insulin resistant by denervation. Available data suggest that malonyl CoA could be linked to insulin resistance in these rodents by virtue of its effects on the cytosolic concentration of long chain fatty acyl CoA (LCFA CoA) and one or more protein kinase C isozymes. Whether similar alterations occur in other tissues and contribute to the pathophysiology of the insulin resistance syndrome remains to be determined.

Photoreceptors are vulnerable in several retinal disorders, including macular degeneration,1 retinal detachment (RD),2–4 diabetic retinopathy,5 retinopathy of prematurity,6 and retinitis pigmentosa.7 In these pathologic conditions, photoreceptors undergo apoptosis.2,5–7 Therefore, new insights about the mechanisms that underlie photoreceptor degeneration in the ocular diseases would be of clinical interest and could lead to new neuroprotective treatments. Previously, we used the rodent model of RD to clarify the mechanism of RD-induced photoreceptor degeneration. We found that RD-induced photoreceptor apoptosis went through a caspase-dependent8,9 or caspase-independent pathway.10 Furthermore, we found that monocytes recruited through the upregulation of monocyte chemoattractant protein (MCP)-1 in Muller glial cells play a neurodestructive role in photoreceptor degeneration.11,12 Tumor necrosis factor (TNF)-α is synthesized, mainly in monocytes, as a 26-kDa precursor13 that is cleaved proteolytically and secreted as a 17-kDa protein.14 TNFα acts via either the low-affinity TNF receptor (TNFR1) or high-affinity TNF receptor (TNFR2).15 TNFα is upregulated in several neurodegenerative disorders including multiple sclerosis, Parkinson's disease, and Alzheimer's disease and suppression of TNFα has demonstrated therapeutic effects.16 In ophthalmic disorders, the vitreous samples from patients with RD contain significantly higher levels of TNFα than samples from patients with other retinal conditions, such as macular hole or idiopathic premacular fibrosis.17,18 However, the role of RD-induced elevated TNFα on photoreceptor degeneration remains unclear. Recently, TNFα-suppressing monoclonal antibodies such as infliximab have been successfully used to treat patients with inflammatory ocular disease, including Behcet's disease,19 diffuse subretinal fibrosis (DSF) syndrome,20 posterior scleritis,21 retinal vascular tumors,22 and neovascular age-related macular degeneration.23 Thus, if TNFα plays a neurodestructive role in RD-induced photoreceptor degeneration, anti-TNFα treatment may be a good candidate for neuroprotective treatment in retinal diseases. In this study, we induced RD in mice deficient in TNF, TNFR1, and TNFR2 and investigated the role of the TNFα pathway on RD-induced photoreceptor apoptosis.

Purpose: Retinoblastoma (Rb) is the most common intraocular tumor in children. Chemotherapy has improved the outcome for both unilateral and bilateral early stage disease, yet late stage bilateral Rb remains difficult to treat, and metastasis are often fatal. Treatment with carboplatin, vincristine and etoposide is only effective in 40% of bilateral Rb patients and results in a high incidence of secondary malignancies. ATP-binding cassette (ABC) transporters act as efflux pumps of different substrates, including drugs. ABCB5, a member of the ABC-B subfamily, is expressed on both normal tissues (CNS, mammary gland, testis, retina) and in skin melanoma, where it identifies a subpopulation of cancer stem cells with enhanced tumorigenicity. Unexpectedly, the retina displays the highest level of ABCG5 in normal tissues, suggesting it might be expressed in Rb tumors. Here we identify for the first time in Rb, an ABCB5+ subpopulation of cells that we predict will possess drug resistance and/or enhanced tumorigenicity. Methods: Rb143, Rb116, Rb125 and Rb107 cell lines were developed in our laboratory from primary explants recovered from patients with large tumors. Sequencing of the Rb gene (27 exons) was used to validate all Rb cell lines. Flow cytometry was used to identify ABCB5+ cells using a monoclonal anti-ABCB5 antibody or isotype control. Calcein AM and/or Aqua fluorescent dye was used to determine viability. ABCB5 pump function was determined by using Calcein AM, a pump substrate that becomes fluorescent within viable cells. Optical coherence tomography (OCT) was used to quantitate the size of intraocular tumors, following orthotopic injection into the sub-retinal space of NOD-scid IL2rg−/− mice. Results: All Rb cell lines possessed a subpopulation of ABCB5+ cells with a frequency ranging from 3-12%. To determine if the ABCB5 pump was functional, Rb cells were cultured for 30 mins with Calcein AM (ABCB5 pump substrate). ABCB5 positive, but not negative, Rb cells excluded the Calcein AM. The ABCB5 pump mediated Calcein exclusion, which was terminated by the addition of an ABCB5 specific blocking antibody. Rb143 cells (originally 3% ABCB5+ cells) were separated by cell sorting into (i) ABCB5+ cells (90% pure), and (ii) ABCB5- cells (100% pure). To test the growth potential and drug resistance of these two subpopulations in vivo, we developed a murine transient retinal detachment model. Injection of 50μl of PBS into the sub-retinal space induces a retinal detachment, but as the PBS is subsequently absorbed over the next 24 hrs the retina reattaches. Up to 1×106 cells were orthotopically injected beneath the detached retina, resulting in tumor growth in < 1 wk. This model will allow us to determine the drug resistance and growth potential of ABCB5+ and ABCB5- Rb cells. Conclusion: Retinoblastoma possesses a small sub-population of ABCB5+ tumor cells, which displays an active pump with the potential of drug resistance and preferential tumor growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4363.

Studies in rats suggest that increases in fatty acid oxidation in skeletal muscle during exercise are related to the phosphorylation and inhibition of acetyl-CoA carboxylase (ACC), and secondary to this, a decrease in the concentration of malonyl-CoA. Studies in human muscle have not revealed a consistent decrease in the concentration of malonyl-CoA during exercise; however, measurements of ACC activity have not been reported. Thus, whether the same mechanism operates in human muscle in response to physical activity remains uncertain. To investigate this question, ACC was immunoprecipitated from muscle of human volunteers and its activity assayed in the same individual at rest and after one-legged knee-extensor exercise at 60, 85, and 100% of knee extensor VO2max. ACC activity was diminished by 50-75% during exercise with the magnitude of the decrease generally paralleling exercise intensity. Treatment of the immunoprecipitated enzyme with protein phosphatase 2A restored activity to resting values, suggesting the decrease in activity was due to phosphorylation. The measurement of malonyl-CoA in the muscles revealed that its concentration is 1/10 of that in rats, and that it is diminished (12-17%) during the higher-intensity exercises. The respiratory exchange ratio increased with increasing exercise intensity from 0.84 +/- 0.02 at 60% to 0.99 0.04 at 100% VO2max. Calculated rates of whole-body fatty acid oxidation were 121 mg/min at rest and 258 +/- 35, 264 +/- 63, and 174 +/- 76 mg/min at 60, 85, and 100% VO2max, respectively. The results show that ACC activity, and to a lesser extent malonyl-CoA concentration, in human skeletal muscle decrease during exercise. Although these changes may contribute to the increases in fat oxidation from rest to exercise, they do not appear to explain the shift from mixed fuel to predominantly carbohydrate utilization when exercise intensity is increased.

PURPOSE. To investigate the anti-inflammatory effect of aminoimidazole carboxamide ribonucleotide (AICAR), an analog of adenosine monophosphate (AMP), in endotoxin-induced uveitis (EIU). METHODS. EIU was induced by subcutaneous injection of lipopolysaccharide (LPS) (200 μg) in Lewis rats. AICAR (50 mg/kg, intraperitoneally) was given 6 hours prior and at the same time as LPS injection. Clinical uveitis scores, number of anterior chamber (AC) infiltrating cells, anterior chamber protein concentration, retinal vessel leukocyte adhesion, and protein leakage were measured 24 hours later. Protein levels of C-C chemokine ligand-2 (CCL-2)/monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNF-α) and intercellular adhesion molecule-1 (ICAM-1) in aqueous humor and retina and nuclear translocation of nuclear factor-κB (NF-κB) in the retina were determined by enzyme-linked immunosorbent assay (ELISA). Both mRNA and protein levels of CD14 in peripheral blood mononuclear cells were also measured. RESULTS. AICAR treatment significantly reduced EIU clinical severity as well as inflammatory cell infiltration and protein concentration in aqueous humor. Similarly, the number of retinal vessel-adherent leukocytes and protein leakage were decreased by AICAR treatment. Protein levels of TNF-α, CCL-2/MCP-1, and ICAM-1 in aqueous humor and CCL-2/MCP-1 and ICAM-1 levels in retina were suppressed with AICAR treatment. AICAR also reduced NF-κB translocation and CD14 expression. CONCLUSIONS. AICAR reduces systemic LPS susceptibility and attenuates intraocular inflammation in a rat EIU model by limiting infiltration of leukocytes, suppressing inflammatory mediators, and inhibiting the NF-κB pathway.

Imaging of the choroidal layer has been limited with the conventional commercial SD-OCTs. Enhanced depth imaging optical coherence tomography (EDI-OCT) is a modification of the standard spectral-domain OCT (SD-OCT) technique that enables better non-invasive imaging of the choroid. This review contains an introduction of EDI imaging technique and principles and summarizes the findings of EDI-OCT imaging in age-related macular degeneration.