The Inhibitory Effect of Blue Light on Phagocytic Activity by ARPE-19 Cells.

Chronic exposure of the retina to short wavelength visible light is a risk factor in pathogenesis of age-related macular degeneration. The proper functioning and survival of photoreceptors depends on efficient phagocytosis of photoreceptor outer segments (POS) by retinal pigment epithelium. The purpose of this study was to analyze the phagocytic activity of blue light-treated ARPE-19 cells, and to examine whether the observed effects could be related to altered levels of POS phagocytosis receptor proteins and/or to oxidation of cellular proteins and lipids. POS phagocytosis was measured by flow cytometry. Phagocytosis receptor proteins αv and ß5 integrin subunits and Mer tyrosine kinase (MerTK) were quantified by western blotting. The intact functional heterodimer αvß5 was quantified by immunoprecipitation followed by immunoblotting. Cellular protein and lipid hydroperoxides were analyzed by coumarin boronic acid probe and iodometric assay, respectively. Cell irradiation induced reversible inhibition of specific phagocytosis and transient reductions in phagocytosis receptor proteins. Full recovery of functional heterodimer was apparent. Significant photooxidation of cellular proteins and lipids was observed. The results indicate that transient inhibition of specific phagocytosis by blue light could be related to the reduction in phagocytosis receptor proteins. Such changes may arise from oxidative modifications of cell phagocytic machinery components.

Evolution of a Revolution in Occupational Therapy Education.

This issue of the American Journal of Occupational Therapy reflects an increase in the quantity and quality of scholarly projects submitted for publication in this special issue. The positive response to the call for papers is evidence of the focused interest that faculty, both academic and clinical, and practitioners have brought to their work of educating and preparing the next generation of occupational therapists. This article discusses educational issues from academic, student, and fieldwork points of view, further validating the pervasive commitment that is being made to further understanding of how occupational therapists are educated. The articles cover topics including the critical issues of curriculum, value and timing of fieldwork and simulation experiences, and documentation of strategies to measure the effects of comparative educational experiences.

Zeaxanthin and α-tocopherol reduce the inhibitory effects of photodynamic stress on phagocytosis by ARPE-19 cells.

Zeaxanthin and α-tocopherol have been previously shown to efficiently protect liposomal membrane lipids against photosensitized peroxidation, and to protect cultured RPE cells against photodynamic killing. Here the protective action of combined zeaxanthin and α-tocopherol was analyzed in ARPE-19 cells subjected to photodynamic (PD) stress mediated by rose Bengal (RB) or merocyanine-540 (MC-540) at sub-lethal levels. Stress-induced cytotoxicity was analyzed by the MTT assay. The peroxidation of membrane lipids was determined by HPLC-EC (Hg) measurements of cholesterol hydroperoxides using cholesterol as a mechanistic reporter molecule. The specific phagocytosis of FITC-labeled photoreceptor outer segments (POS) isolated from bovine retinas was measured by flow cytometry, and the levels of phagocytosis receptor proteins αv integrin subunit, ß5 integrin subunit and MerTK were quantified by Western blot analysis. Cytotoxicity measures confirmed that PD stress levels used for phagocytosis analysis were sub-lethal and that antioxidant supplementation protected against higher, lethal PD doses. Sub-lethal PD stress mediated by both photosensitizers induced the accumulation of 5α-OOH and 7α/ß-OOH cholesterol hydroperoxides and the addition of the antioxidants substantially inhibited their accumulation. Antioxidant delivery prior to PD stress also reduced the inhibitory effect of stress on POS phagocytosis and partially reduced the stress-induced diminution of phagocytosis receptor proteins. The use of a novel model system where oxidative stress was induced at sub-lethal levels enable observations that would not be detectable using lethal stress models. Moreover, novel observations about the protective effects of zeaxanthin and α-tocopherol on photodynamic damage to ARPE-19 cell membranes and against reductions in the abundance of receptor proteins involved in POS phagocytosis, a process essential for photoreceptor survival, supports the importance of the antioxidants in protecting of the retina against photooxidative injury.

Linking Sensory Factors to Participation: Establishing Intervention Goals With Parents for Children With Autism Spectrum Disorder.

Parents often focus on independence in activities of daily living and social participation when setting goals for their children with autism spectrum disorders. Occupational therapy practitioners use clinical reasoning to translate these goals to define occupation-based outcomes. This article describes an exploratory analysis of 160 parent-identified goals for children with autism. We identified sensory integrative factors hypothesized to influence each goal and then categorized the goals using the Occupational Therapy Practice Framework: Domain and Process and the International Classification of Functioning, Disability and Health (ICF). Most goals were at the ICF participation and activity levels. Activities of daily living were the most common area of occupation identified, followed by social participation and play. Sensory reactivity and somatopraxis were the most frequently occurring sensory integrative factors. The value of addressing parent goals using a systematic reasoning process to identify factors affecting participation and the importance of measuring participation outcomes are discussed.

Scholarship of Practice in the Care of People with Dementia: Creating the Future Through Collaborative Efforts.

A scholarship of practice approach sets the stage for collaborative partnerships across academic and clinical practice settings that result in positive gains for all stakeholders. These gains include an enhanced ability to generate and apply relevant evidence in practice, disseminate knowledge and innovation, and ensure best practice is relevant to and effective for, people receiving services and their caregivers. This paper discusses national and international examples of collaborative, research-based practice initiatives that have implemented a scholarship of practice approach. The exemplars described here are framed within the Model of Human Occupation, which addresses the importance of volition, habits, roles, environment, and performance capacities in facilitating engagement in occupation for people with dementia. Research that focuses on how therapists adopt and use evidence in practice, as well as the opportunities and challenges for supporting therapists and their use of theory and evidence are discussed.

State of measurement in occupational therapy using sensory integration.

This article presents the current state of measurement in the area of sensory integration within the field of occupational therapy in three areas: (1) phenotypic characterization, (2) intervention adherence and dosage, and (3) outcome measurement. The need for additional measurement tools in all three areas is addressed. In regard to outcome measurement of occupational therapy using sensory integration, the use of both qualitative and quantitative methods to obtain outcome data is recommended. Further, a strategy is recommended for obtaining outcome data from direct report from the child or other stakeholder.

Photic injury to cultured RPE varies among individual cells in proportion to their endogenous lipofuscin content as modulated by their melanosome content.

PURPOSE: We determined whether photic stress differentially impairs organelle motility of RPE lipofuscin and melanin granules, whether lethal photic stress kills cells in proportion to lipofuscin abundance, and whether killing is modulated by melanosome content. METHODS: Motility of endogenous lipofuscin and melanosome granules within the same human RPE cells in primary culture was quantified by real-time imaging during sublethal blue light irradiation. Cell death during lethal irradiation was quantified by dynamic imaging of the onset of nuclear propidium iodide fluorescence. Analyzed were individual cells containing different amounts of autofluorescent lipofuscin, or similar amounts of lipofuscin and a varying content of phagocytized porcine melanosomes, or phagocytized black latex beads (control for light absorbance). RESULTS: Lipofuscin granules and melanosomes showed motility slowing with mild irradiation, but slowing was greater for lipofuscin. On lethal irradiation, cell death was earlier in cells with higher lipofuscin content, but delayed by the copresence of melanosomes. Delayed death did not occur with black beads, suggesting that melanosome protection was due to properties of the biological granule, not simple screening. CONCLUSIONS: Greater organelle motility slowing of the more photoreactive lipofuscin granule compared to melanosomes suggests that lipofuscin mediates mild photic injury within RPE cells. With lethal light stress endogenous lipofuscin mediates killing, but the effect is cell autonomous and modulated by coincident melanosome content. Developing methods to quantify the frequency of individual cells with combined high lipofuscin and low melanosome content may have value for predicting the photic stress susceptibility of the RPE monolayer in situ.

Photosensitized oxidative stress to ARPE-19 cells decreases protein receptors that mediate photoreceptor outer segment phagocytosis.

PURPOSE: To determine whether previously shown photodynamic (PD)-induced inhibition of specific photoreceptor outer segment (POS) phagocytosis by ARPE-19 cells is associated with reductions in receptor proteins mediating POS phagocytosis, and if PD treatment with merocyanine-540 (MC-540) produces additional effects leading to its inhibition of nonspecific phagocytosis. METHODS: ARPE-19 cells preloaded with MC-540 or rose bengal (RB) were sublethally irradiated with green light. Phagocytosis of POS was measured by flow cytometry and POS receptor proteins (Mer tyrosine kinase receptor [MerTK] and integrin subunits αv and ß5) and ß-actin were quantified by Western blotting at 0.5 and 24 hours after irradiation, with comparison to samples from nonsensitized control cultures. The intact integrin heterodimer αvß5 was quantified by immunoprecipitation followed by blotting. The distribution of N-cadherin, ZO-1, and F-actin was visualized by fluorescence microscopy. RESULTS: Mild PD stress mediated by both photosensitizers that elicits no significant morphologic changes produces transient and recoverable reductions in MerTK. The individual αv and ß5 integrin subunits are also reduced but only partially recover. However, there is sufficient recovery to support full recovery of the functional heterodimer. Light stress mediated by MC-540 also reduced levels of actin, which is known to participate in the internalization of particles regardless of type. CONCLUSIONS: After PD treatment POS receptor protein abundance and phagocytosis show a coincident in time reduction then recovery suggesting that diminution in receptor proteins contributes to the phagocytic defect. The additional inhibition of nonspecific phagocytosis by MC-540-mediated stress may result from more widespread effects on cytosolic proteins. The data imply that phagocytosis receptors in RPE cells are sensitive to oxidative modification, raising the possibility that chronic oxidative stress in situ may reduce the efficiency of the RPE's role in photoreceptor turnover, thereby contributing to retinal degenerations.

Dissemination: bringing translational research to completion.

Despite the availability of innovative health care research, a gap exists between research-generated knowledge and the utilization of that knowledge in real-world practice settings. This article examines the transition from research to implementation in the context of the dissemination of A. Jean Ayres' sensory integration procedures and of the challenges currently facing the University of Southern California Well Elderly Studies research team. Drawing from the emerging field of implementation science, this article discusses how researchers can develop an implementation plan to more easily translate evidence into practice. Such plans should address the intervention's reach (i.e., its capacity to penetrate into the intended target population), the settings for which it is applicable, the leaders who will encourage practitioner uptake, stakeholder groups, and challenges to dissemination. By taking action to ensure the more effective dissemination of research-generated knowledge, researchers can increase the likelihood that their interventions will lead to improvements in practice and more effective care for consumers.

Photoaging of human retinal pigment epithelium is accompanied by oxidative modifications of its eumelanin.

Although photodegradation of the retinal pigment epithelium (RPE) melanin may contribute to the etiology of age-related macular degeneration, the molecular mechanisms of this phenomenon and the structural changes of the modified melanin remain unknown. Recently, we found that the ratio of pyrrole-2,3,4,5-tetracarboxylic acid (PTeCA) to pyrrole-2,3,5-tricarboxylic acid (PTCA) is a marker for the heat-induced cross-linking of eumelanin. In this study, we examined UVA-induced changes in synthetic eumelanins to confirm the usefulness of the PTeCA/PTCA ratio as an indicator of photo-oxidation and compared changes in various melanin markers and their ratios in human melanocytes exposed to UVA, in isolated bovine RPE melanosomes exposed to strong blue light and in human RPE cells from donors of various ages. The results indicate that the PTeCA/PTCA ratio is a sensitive marker for the oxidation of eumelanin exposed to UVA or blue light and that eumelanin and pheomelanin in human RPE cells undergo extensive structural modifications due to the life-long exposure to blue light.

Oxidative stress increases HO-1 expression in ARPE-19 cells, but melanosomes suppress the increase when light is the stressor.

PURPOSE: Phagocytized melanosomes in ARPE-19 cells were previously shown to decrease susceptibility to oxidative stress induced by hydrogen peroxide treatment and increase stress due to light irradiation relative to cells containing control black latex beads. Here we asked whether differential expression of antioxidant enzymes in cells containing pigment granules could explain the outcomes. METHODS: ARPE-19 cells were loaded by phagocytosis with porcine RPE melanosomes or black latex beads (control particles). Heme oxygenase-1 (HO-1), HO-2, glutathione peroxidase (GPx), and catalase were quantified by Western blot analysis before and after treatment with sublethal hydrogen peroxide or blue light (400-450 nm). The stress was confirmed as sublethal by cell survival analysis using real-time quantification of propidium iodide fluorescence. RESULTS: Phagocytosis itself produced transient changes in protein levels of some antioxidant enzymes, but steady-state levels (7 days after phagocytosis) did not differ in cells containing melanosomes versus beads. Sublethal stress, induced by either hydrogen peroxide or light, had no effect on catalase or HO-2 in either particle-free or particle-loaded cells. In contrast, HO-1 protein was upregulated by treatment with both hydrogen peroxide and light. Particle content did not affect the HO-1 increase induced by hydrogen peroxide, but the increase induced by blue light irradiation was partially blocked in cells containing black beads and blocked even more in cells containing melanosomes. CONCLUSIONS: The results do not implicate differential antioxidant enzyme levels in stress protection by melanosomes against hydrogen peroxide, but they suggest a multifaceted role for melanosomes in regulating light stress susceptibility in RPE cells.

Melanosome-iron interactions within retinal pigment epithelium-derived cells.

Melanosomes were recently shown to protect ARPE-19 cells, a human retinal pigment epithelium (RPE) cell line, against oxidative stress induced by hydrogen peroxide. One postulated mechanism of antioxidant action of melanin is its ability to bind metal ions. The aim here was to determine whether melanosomes are competent to bind iron within living cells, exhibiting a property previously shown only in model systems. The outcomes indicate retention of prebound iron and accumulation of iron by granules after iron delivery to cells via the culture medium, as determined by both colorimetric and electron spin resonance analyses for bound-to-melanosome iron. Manipulation of iron content did not affect the pigment's ability to protect cells against H(2) O(2) , but the function of pigment granules within RPE cells should be extended beyond a role in light irradiation to include participation in iron homeostasis.

How do we change practice when we have the evidence?

Translating research findings into practice includes myriad pragmatic realities, including understanding the suitability of the data to a particular patient group, writing new guidelines for occupational therapy practitioners, facilitating adoption of the guidelines, and instituting new patterns of care for patients. The process is more than a matter of disseminating the information to practitioners and expecting immediate change in patient treatment. Indeed, the field of implementation science is devoted to the identification of the numerous barriers and supports that constrain or expedite practice change in response to research. Moving forward and adopting evidence-based findings will require a focused understanding of the particular setting where change is warranted. Among the issues to address are the health system levels involved in change (professional, legislative, administrative, practitioner, and patient and family members), the values and beliefs of the participants, and knowledge of the communication channels that exist in the setting and how information and new ideas make their way through the setting.

Integrating photoacoustic ophthalmoscopy with scanning laser ophthalmoscopy, optical coherence tomography, and fluorescein angiography for a multimodal retinal imaging platform.

Photoacoustic ophthalmoscopy (PAOM) is a newly developed retinal imaging technology that holds promise for both fundamental investigation and clinical diagnosis of several blinding diseases. Hence, integrating PAOM with other existing ophthalmic imaging modalities is important to identify and verify the strengths of PAOM compared with the established technologies and to provide the foundation for more comprehensive multimodal imaging. To this end, we developed a retinal imaging platform integrating PAOM with scanning laser ophthalmoscopy (SLO), spectral-domain optical coherence tomography (SD-OCT), and fluorescein angiography (FA). In the system, all the imaging modalities shared the same optical scanning and delivery mechanisms, which enabled registered retinal imaging from all the modalities. High-resolution PAOM, SD-OCT, SLO, and FA images were acquired in both albino and pigmented rat eyes. The reported in vivo results demonstrate the capability of the integrated system to provide comprehensive anatomic imaging based on multiple optical contrasts.

Near-infrared light photoacoustic ophthalmoscopy.

We achieved photoacoustic ophthalmoscopy (PAOM) imaging of the retina with near-infrared (NIR) light illumination. A PAOM imaging system with dual-wavelength illumination at 1064 nm and 532 nm was built. We compared in vivo imaging results of both albino and pigmented rat eyes at the two wavelengths. The results show that the bulk optical absorption of the retinal pigment epithelium (RPE) is only slightly higher than that of the retinal vessels at 532 nm while it becomes more than an order of magnitude higher than that of the retinal vessels at 1064 nm. These studies suggest that although visible light illumination is suitable for imaging both the retinal vessels and the RPE, NIR light illumination, being more comfortable to the eye, is better suited for RPE melanin related investigations and diagnoses.

Dynamic analyses reveal cytoprotection by RPE melanosomes against non-photic stress.

PURPOSE: Isolated melanosomes are known to have antioxidant properties but whether the granules perform an antioxidant function within cells is unclear. The aim of this study was to determine whether retinal pigment epithelium (RPE) melanosomes are competent to protect cultured cells against non-photic oxidative stress induced by treatment with H(2)O(2). METHODS: Porcine melanosomes, either untreated or irradiated with visible light to simulate age-related melanin photobleaching, were introduced by phagocytosis into ARPE-19 cells. Cells were treated with H(2)O(2) using two delivery methods: as a pulse, or by continuous generation following addition of glucose oxidase to the medium. Cell survival in melanosome-containing cells was compared to survival in cells containing phagocytosed control latex beads using two real-time cell death assays. RESULTS: Following H(2)O(2) delivery by either method, greater resistance to critical concentrations of H(2)O(2) was seen for cells containing melanosomes than for cells containing beads. Melanosome-mediated protection manifested as a delay in the time of onset of cell death and a slower rate of cell death over time. Photobleaching diminished the stress resistance conferred by the pigment granules. Individual cells in co-cultures were differentially sensitive to oxidative stress depending upon their particle content. Additional features of the time course of the cell death response were revealed by the dynamic analyses conducted over hours post oxidant treatment. CONCLUSIONS: The results show, for the first time, that melanosomes perform a cytoprotective function within cultured cells by acting as an antioxidant. The outcomes imply that melanosomes perform functions within RPE cells aside from those related to light irradiation, and also suggest that susceptibility to ubiquitous pro-oxidizing agents like H(2)O(2) is partly determined by discrete features of individual RPE cells such as their granule content.

Raman microspectroscopy of melanosomes: the effect of long term light irradiation.

Melanosomes are long lived organelles in retinal pigment epithelium cells and are primarily responsible for photoprotection. However, with aging or prolong light radiation, the function of melanosomes diminishes, which may be due to photobleaching of melanin pigments present in melanosomes. In this study, melanosomes were isolated from the retinal pigment epithelium cells and exposed to green light (532 nm), and the chemical changes were monitored using Raman microspectroscopy. Photochemical changes were recorded for different power levels and exposure times. The threshold power and the rate for irreversible photobleaching of melanosomes were calculated by fitting the experimental data with a proposed model.

Development of a fidelity measure for research on the effectiveness of the Ayres Sensory Integration intervention.

OBJECTIVE: We developed a reliable and valid fidelity measure for use in research on Ayres Sensory Integration (ASI) intervention. METHOD: We designed a fidelity instrument to measure structural and process aspects of ASI intervention. Because scoring of process involves subjectivity, we conducted a series of reliability and validity studies on the process section. Raters were trained to score therapist strategies observed in video recordings of adult-child dyads. We examined content validity through expert ratings. RESULTS: Reliability of the process section was strong for total fidelity score (ICC = .99, Cronbach's alpha = .99) and acceptable for most items. Total score significantly differentiated ASI from four alternative interventions. Expert ratings indicated strong agreement that items in the structural and process sections represent ASI intervention. CONCLUSION. The Ayres Sensory Integration Fidelity Measure has strong content validity. The process section is reliable and valid when scored by trained raters with expertise in ASI.

Evaluating verbal and non-verbal communication skills, in an ethnogeriatric OSCE.

OBJECTIVE: Communication during medical interviews plays a large role in patient adherence, satisfaction with care, and health outcomes. Both verbal and non-verbal communication (NVC) skills are central to the development of rapport between patients and healthcare professionals. The purpose of this study was to assess the role of non-verbal and verbal communication skills on evaluations by standardized patients during an ethnogeriatric Objective Structured Clinical Examination (OSCE). METHODS: Interviews from 19 medical students, residents, and fellows in an ethnogeriatric OSCE were analyzed. Each interview was videotaped and evaluated on a 14 item verbal and an 8 item non-verbal communication checklist. The relationship between verbal and non-verbal communication skills on interview evaluations by standardized patients were examined using correlational analyses. RESULTS: Maintaining adequate facial expression (FE), using affirmative gestures (AG), and limiting both unpurposive movements (UM) and hand gestures (HG) had a significant positive effect on perception of interview quality during this OSCE. Non-verbal communication skills played a role in perception of overall interview quality as well as perception of culturally competent communication. CONCLUSION: Incorporating formative and summative evaluation of both verbal and non-verbal communication skills may be a critical component of curricular innovations in ethnogeriatrics, such as the OSCE.