Natural History of Visual Acuity and Microperimetry-Based Functional Outcome Measures of the Macula in Patients with Geographic Atrophy: A Retrospective Chart Review Study in Germany.

Background/Objectives: Geographic atrophy (GA) is an advanced form of age-related macular degeneration (AMD) leading to the progressive and irreversible loss of visual function. Characteristics of GA include atrophic lesions resulting from the loss of photoreceptors, retinal pigment epithelium, and choriocapillaris. During GA progression, atrophic lesions typically advance from the macular periphery to the center, affecting foveal light sensitivity and visual acuity. This study analyzed changes in light sensitivity and visual acuity during the natural course of GA progression using the topographic analysis of structural and functional changes based on Early Treatment Diabetic Retinopathy Study (ETDRS) charts, multimodal imaging, and microperimetry assessment. Methods: Medical chart data of GA patients between 2014 and 2022 from the Internationale Innovative Ophthalmochirurgie GbR (I.I.O.) research center (Düsseldorf, Germany) were retrospectively analyzed. All patient eyes fulfilling the phase 3 OAKS study inclusion criteria were included and followed up for 60 months. The imputation of missing measurements and dropouts was performed by linear mixed models. Results: A total of 20 GA eyes from 13 GA patients were included in the study. At the index, 53.8% of patients had bilateral GA, with 70.0% of the eyes showing multifocal GA and 30.0% subfoveal encroachment (SFE). A total of 35.0% of the eyes had 2-5, and 15.0% over 20, areas of atrophy. Over time, the GA lesion size increased from 6.4 mm2 to 11.8 mm2 (1.08 mm2/year). After an average observation time of 2.9 years, 78.6% of the initially unaffected study eyes developed SFE. The percentage of study eyes without visual impairment decreased from 55.0% to 30.0%, with mean normal-luminance best-corrected visual acuity (NL-BCVA) reducing from 63.7 to 55.7 ETDRS letters. The share of absolute scotoma points in microperimetry assessment increased from 15.7% to 43.5% while overall average macular sensitivity declined from 15.7 dB to 7.4 dB. Conclusions: The substantial deterioration of macular outcomes and visual function was comprehensively detected. The results were a documentation of structural and functional aspects of the natural progression of GA for a 60-month follow-up, providing a typical outline for AMD patients with GA.

Compensation of adverse growing media effects on plant growth and morphology by supplemental LED lighting.

There is an increasing interest in alternatives to peat in growing media due to environmental constraints. However, plants grown in peat substitutes often show impaired growth compared to plants grown in peat-based media. Hence, it would be interesting to know whether these deficiencies can be compensated by supplementing other growth factors, e.g. light. The present study aims to investigate the interactive nature between growing media and supplemental lighting on plant growth and morphology, and to examine whether supplemental light emitting diode (LED) lighting may compensate adverse growing media effects. Basil (Ocimum basilicum L.) and Chinese cabbage (Brassica rapa subsp. pekinensis) were grown in different growing media consisting of peat, green compost, coconut pulp, wood fibre, perlite and sphagnum moss under blue, red and far-red supplemental LED lighting. We found significant interactions between growing media and supplemental photosynthetically active radiation (PAR) on plant growth, morphology and development. At low light intensities, peat-based and substituted growing media performed similarly, whereas with increasing light intensities the peat-based growing media significantly outperformed their alternatives. The substrate choice determines the required amount of supplemental light to compensate for adverse growing media effects and the amount varies depending on plant species and season. Thereby, it was indicated that red light alleviates adverse growing media effects best. We also found that far-red light is not effective when background PAR is low and becomes more effective under high background PAR. The implications and prospects of the results are discussed.

Seasonal Efficiency of Supplemental LED Lighting on Growth and Photomorphogenesis of Sweet Basil.

For decisions on supplemental lighting a quantitative knowledge of the plants' responses to light under varying conditions is fundamental. In this study, we developed light dose-response curves of growth and morphological traits for Ocimum basilicum L. and examined the effects of light color (blue, red, and white plus far-red) and natural environment (season) on these curves. Four greenhouse experiments were conducted throughout the year to determine the efficiencies of the light regimes on growth and their effects on plant morphology. A special aspect was the photosynthetic efficiency of far-red light. Linear and monomolecular relationships were found for the relationships between plant traits and supplemental light dose. Traits related to biomass productivity increased linearly with light dose whereas some morphological characters showed a saturation behavior. Red light and white plus far-red light were more efficient in plant dry weight production than blue light, and the plants adapted differently to the light qualities: higher biomass under red light was related to a plant architecture more favorable for light capture, i.e., taller plants and bigger leaves. White plus far-red light, on the other hand, increased leaf mass per area (LMA) and light use efficiency (LUE). Blue light resulted in lowest plant light interception and LUE. Considering photosynthetic effects of near-infrared light (PPFD800, 400-800 nm) instead of photosynthetic photon flux density (PPFD700, 400-700 nm) led to strongly reduced efficiencies. Traits related to photosynthesis such as dry weight, LMA and LUE were particularly affected by PPFD800. There were no interactions between the efficiencies of the different light colors and the seasons. Efficiencies of all light regimes were significantly lower during summer compared to spring and winter. Higher dry weight production during summer compared to winter and spring were a consequence of increased light interception rather than changes in LUE. The observed differences in seasonal efficiencies were directly linked to the amount of natural light present as indicated by changes in the ratio of supplemental to natural light.

Efficacy of nanosecond laser treatment in central serous chorioretinopathy with and without atrophy of retinal pigment epithelium.

BACKGROUND: To evaluate the outcomes of subthreshold nanosecond laser treatment of chronic central serous chorioretinopathy (CSC) as a function of the severity of concomitant of retinal pigment epithelium (RPE) defects. METHODS: This retrospective study compares data from 23 CSC diagnosed eyes with only mild RPE defects (group 1), 16 CSC eyes with moderate RPE defects (group 2), and 17 CSC eyes having severe RPE defects (group 3). After subthreshold treatment with the standard Ellex 2RT™ nanosecond laser (Ellex Medical Lasers Ltd, Australia), changes in macular structure and levels of subretinal fluid (SRF) were assessed by OCT-SD, OCT-A, functional integrity of the retina was assessed by corrected distance visual acuity (CDVA) and microperimetry, each at baseline and 1, 3, 6, and 12 months after initial treatment; re-treatment took place in cases of persistent SRF pro re nata. RESULTS: During the 12 months observation period, group 1 and 2 mostly required on initial and one re-treatment (1.9 ± 1.0 treatments; 1.9 ± 1.3 treatments). In contrast, group 3 was subject to three to four treatments (3.7 ± 1.5 treatments). 6 to 12 months after treatment, subretinal fluid (SRF) disappeared in 100% of the eyes of group 1 and in 76.9%, and 42.9% of the eyes of group 2 and group 3, respectively. Retinal sensitivity and CDVA improved in group 1 and 2, but did not change significantly in group 3 during the 12 months period. CONCLUSIONS: Subthreshold nanosecond laser treatment is an effective and safe method for the restoration of macular anatomy and sensitivity in acute and chronic CSC cases with only mild or moderate RPE defects. However, this laser treatment has very limited outcome in CSC eyes with more severe RPE defects.

Short-term effects of low-concentration atropine eye drops on pupil size and accommodation in young adult subjects.

PURPOSE: A single eye drop containing 0.01% atropine every evening has previously been found to inhibit myopia progression in young adults. We have tested the short-term effects of very low-dose atropine eye drops on pupil sizes and accommodation in young adult subjects. METHODS: Fourteen eyes of young adult subjects participated in the clinical observation. A single eye drop was applied with concentrations of either 0.01%, 0.005%, or 0.001% in the evening. Baseline parameters were measured before atropine application. Changes of pupil sizes, under photopic and mesopic conditions, as well as accommodation amplitudes were observed over the next day and analyzed by paired the Wilcoxon signed-rank test. RESULTS: The pupil was significantly dilated 12 h after instillation of 0.01% atropine eye drops, both under photopic (3.3 ± 0.5 mm vs. 4.9 ± 0.9 mm) and mesopic (4.8 ± 0.7 mm vs. 6.1 ± 0.7 mm) conditions. Pupil sizes recovered over the day but were still significantly larger in the evening, compared to the baseline parameters measured on the day before (3.9 ± 0.5 mm vs. 5.3 ± 0.6 mm). The subjective near point of accommodation was reduced from 8.0 ± 2.4 to 6.6 ± 2.8 dpt in the morning and to 7.0 ± 2.9 dpt in the evening. At 0.005%, the pattern of results remained still similar, although the magnitude of the effects was generally smaller. At 0.001%, pupil sizes were still weakly significantly larger in the morning. CONCLUSIONS: At a dose of 0.01%, clinically significant short-term effects were detected on pupil size and accommodation for at least 24 h. At the lowest dose of 0.001%, only tiny effects on pupil size were detectable.

Accumulation of DNA damage in hematopoietic stem and progenitor cells during human aging.

BACKGROUND: Accumulation of DNA damage leading to adult stem cell exhaustion has been proposed to be a principal mechanism of aging. Here we tested this hypothesis in healthy individuals of different ages by examining unrepaired DNA double-strand breaks (DSBs) in hematopoietic stem/progenitor cells matured in their physiological microenvironment. METHODOLOGY/PRINCIPAL FINDINGS: To asses DNA damage accumulation and repair capacities, γH2AX-foci were examined before and after exposure to ionizing irradiation. Analyzing CD34+ and CD34- stem/progenitor cells we observed an increase of endogenous γH2AX-foci levels with advancing donor age, associated with an age-related decline in telomere length. Using combined immunofluorescence and telomere-fluorescence in-situ hybridization we show that γH2AX-foci co-localize consistently with other repair factors such as pATM, MDC1 and 53BP1, but not significantly with telomeres, strongly supporting the telomere-independent origin for the majority of foci. The highest inter-individual variations for non-telomeric DNA damage were observed in middle-aged donors, whereas the individual DSB repair capacity appears to determine the extent of DNA damage accrual. However, analyzing different stem/progenitor subpopulations obtained from healthy elderly (>70 years), we observed an only modest increase in DNA damage accrual, most pronounced in the primitive CD34+CD38(-)-enriched subfraction, but sustained DNA repair efficiencies, suggesting that healthy lifestyle may slow down the natural aging process. CONCLUSIONS/SIGNIFICANCE: Based on these findings we conclude that age-related non-telomeric DNA damage accrual accompanies physiological stem cell aging in humans. Moreover, aging may alter the functional capacity of human stem cells to repair DSBs, thereby deteriorating an important genome protection mechanism leading to exceeding DNA damage accumulation. However, the great inter-individual variations in middle-aged individuals suggest that additional cell-intrinsic mechanisms and/or extrinsic factors contribute to the age-associated DNA damage accumulation.

The impact of individual in vivo repair of DNA double-strand breaks on oral mucositis in adjuvant radiotherapy of head-and-neck cancer.

PURPOSE: To evaluate the impact of individual in vivo DNA double-strand break (DSB) repair capacity on the incidence of severe oral mucositis in patients with head-and-neck cancer undergoing adjuvant radiotherapy (RT) or radiochemotherapy (RCT). PATIENTS AND METHODS: Thirty-one patients with resected head-and-neck cancer undergoing adjuvant RT or RCT were examined. Patients underwent RT of the primary tumor site and locoregional lymph nodes with a total dose of 60-66 Gy (single dose 2 Gy, five fractions per week). Chemotherapy consisted of two cycles of cisplatin and 5-fluorouracil. To assess DSB repair, γ-H2AX foci in blood lymphocytes were quantified before and 0.5 h, 2.5 h, 5 h, and 24 h after in vivo radiation exposure (the first fraction of RT). World Health Organization scores for oral mucositis were documented weekly and correlated with DSB repair. RESULTS: Sixteen patients received RT alone; 15 patients received RCT. In patients who developed Grade≥3 mucositis (n=18) the amount of unrepaired DSBs 24 h after radiation exposure and DSB repair half-times did not differ significantly from patients with Grade≤2 mucositis (n=13). Patients with a proportion of unrepaired DSBs after 24 h higher than the mean value + one standard deviation had an increased incidence of severe oral mucositis. CONCLUSIONS: Evaluation of in vivo DSB repair by determination of γ-H2AX foci loss is feasible in clinical practice and allows identification of patients with impaired DSB repair. The incidence of oral mucositis is not closely correlated with DSB repair under the evaluated conditions.

Protecting the heritable genome: DNA damage response mechanisms in spermatogonial stem cells.

Spermatogonial stem cells (SSCs) must maintain the integrity of their genome to prevent reproduction failure and limit the hereditary risk associated with transmission to the progeny. SSCs must therefore have robust response mechanisms to counteract the potentially deleterious effects of DNA damage, with DNA double-strand breaks (DSBs) representing the greatest threat to genomic integrity. Through in vivo analysis of the DNA damage response of SSCs within their physiological tissue context, we aimed to gain insights into the mechanisms by which SSCs preserve genome integrity. After whole-body irradiation of repair-proficient and repair-deficient (DNA-PK- and ATM-deficient) mice, the formation and rejoining of DSBs was analyzed in SSCs of testis compared with somatic cells of other tissues by enumerating γH2AX-, MDC1-, and 53BP1-foci. Caspase-3 and PARP-1 were used as markers for apoptotic cell death. Our results show that DNA damage response mechanisms in SSCs characterized by unique chromatin compositions are markedly different from those of somatic cells. In SSCs lacking compact heterochromatin, histone-associated signaling components of the DNA repair machinery are completely absent and radiation-induced DSBs are rejoined predominantly by DNA-PK-independent pathways, suggesting the existence of alternative repair mechanisms. As a complimentary mechanism characterized by low thresholds for ATM-dependent checkpoint activation, the differentiating progeny, but not the SSCs themselves, promote apoptosis in response to low levels of DNA damage. By evaluating SSCs within their stem cell niche, we show that DNA repair, cell-cycle checkpoints, and apoptosis function together to maintain the integrity of the heritable genome.

Accumulation of DNA double-strand breaks in normal tissues after fractionated irradiation.

PURPOSE: There is increasing evidence that genetic factors regulating the recognition and/or repair of DNA double-strand breaks (DSBs) are responsible for differences in radiosensitivity among patients. Genetically defined DSB repair capacities are supposed to determine patients' individual susceptibility to develop adverse normal tissue reactions after radiotherapy. In a preclinical murine model, we analyzed the impact of different DSB repair capacities on the cumulative DNA damage in normal tissues during the course of fractionated irradiation. MATERIAL AND METHODS: Different strains of mice with defined genetic backgrounds (SCID(-/-) homozygous, ATM(-/-) homozygous, ATM(+/-)heterozygous, and ATM(+/+)wild-type mice) were subjected to single (2 Gy) or fractionated irradiation (5 x 2 Gy). By enumerating gammaH2AX foci, the formation and rejoining of DSBs were analyzed in organs representative of both early-responding (small intestine) and late-responding tissues (lung, kidney, and heart). RESULTS: In repair-deficient SCID(-/-) and ATM(-/-)homozygous mice, large proportions of radiation-induced DSBs remained unrepaired after each fraction, leading to the pronounced accumulation of residual DNA damage after fractionated irradiation, similarly visible in early- and late-responding tissues. The slight DSB repair impairment of ATM(+/-)heterozygous mice was not detectable after single-dose irradiation but resulted in a significant increase in unrepaired DSBs during the fractionated irradiation scheme. CONCLUSIONS: Radiation-induced DSBs accumulate similarly in acute- and late-responding tissues during fractionated irradiation, whereas the whole extent of residual DNA damage depends decisively on the underlying genetically defined DSB repair capacity. Moreover, our data indicate that even minor impairments in DSB repair lead to exceeding DNA damage accumulation during fractionated irradiation and thus may have a significant impact on normal tissue responses in clinical radiotherapy.

DNA repair alterations in children with pediatric malignancies: novel opportunities to identify patients at risk for high-grade toxicities.

PURPOSE: To evaluate, in a pilot study, the phosphorylated H2AX (γH2AX) foci approach for identifying patients with double-strand break (DSB) repair deficiencies, who may overreact to DNA-damaging cancer therapy. METHODS AND MATERIALS: The DSB repair capacity of children with solid cancers was analyzed compared with that of age-matched control children and correlated with treatment-related normal-tissue responses (n = 47). Double-strand break repair was investigated by counting γH2AX foci in blood lymphocytes at defined time points after irradiation of blood samples. RESULTS: Whereas all healthy control children exhibited proficient DSB repair, 3 children with tumors revealed clearly impaired DSB repair capacities, and 2 of these repair-deficient children developed life-threatening or even lethal normal-tissue toxicities. The underlying mutations affecting regulatory factors involved in DNA repair pathways were identified. Moreover, significant differences in mean DSB repair capacity were observed between children with tumors and control children, suggesting that childhood cancer is based on genetic alterations affecting DSB repair function. CONCLUSIONS: Double-strand break repair alteration in children may predispose to cancer formation and may affect children's susceptibility to normal-tissue toxicities. Phosphorylated H2AX analysis of blood samples allows one to detect DSB repair deficiencies and thus enables identification of children at risk for high-grade toxicities.

HDAC inhibition radiosensitizes human normal tissue cells and reduces DNA Double-Strand Break repair capacity.

HDAC inhibitors (HDACi) are gaining increasing attention in the treatment of cancer, particularly in view of their therapeutic effectiveness and assumed mild toxicity profile. While numerous studies have investigated the role of HDACi in tumor cells, little is known about their effects on normal tissue cells. We studied the effect of suberoylanilide hydroxamic acid (SAHA), MS275, sodium-butyrate and valproic acid in healthy human fibroblasts and found HDACi-treatment to go along with increased radiosensitivity and reduced DSB repair capacity. In view of the potential genotoxic effects of HDACi-treatment, particularly when being administered long-term for chronic disease or when given to children, to women of childbearing age or their partners or in combination with radiotherapy, an extensive education of patients and prescribing physicians as well as a stringent definition of clinical indications is urgently required.

DNA double-strand break rejoining in complex normal tissues.

PURPOSE: The clinical radiation responses of different organs vary widely and likely depend on the intrinsic radiosensitivities of their different cell populations. Double-strand breaks (DSBs) are the most deleterious form of DNA damage induced by ionizing radiation, and the cells' capacity to rejoin radiation-induced DSBs is known to affect their intrinsic radiosensitivity. To date, only little is known about the induction and processing of radiation-induced DSBs in complex normal tissues. Using an in vivo model with repair-proficient mice, the highly sensitive gammaH2AX immunofluorescence was established to investigate whether differences in DSB rejoining could account for the substantial differences in clinical radiosensitivity observed among normal tissues. METHODS AND MATERIALS: After whole body irradiation of C57BL/6 mice (0.1, 0.5, 1.0, and 2.0 Gy), the formation and rejoining of DSBs was analyzed by enumerating gammaH2AX foci in various organs representative of both early-responding (small intestine) and late-responding (lung, brain, heart, kidney) tissues. RESULTS: The linear dose correlation observed in all analyzed tissues indicated that gammaH2AX immunofluorescence allows for the accurate quantification of DSBs in complex organs. Strikingly, the various normal tissues exhibited identical kinetics for gammaH2AX foci loss, despite their clearly different clinical radiation responses. CONCLUSION: The identical kinetics of DSB rejoining measured in different organs suggest that tissue-specific differences in radiation responses are independent of DSB rejoining. This finding emphasizes the fundamental role of DSB repair in maintaining genomic integrity, thereby contributing to cellular viability and functionality and, thus, tissue homeostasis.

A pathway of double-strand break rejoining dependent upon ATM, Artemis, and proteins locating to gamma-H2AX foci.

The hereditary disorder ataxia telangiectasia (A-T) is associated with striking cellular radiosensitivity that cannot be attributed to the characterized cell cycle checkpoint defects. By epistasis analysis, we show that ataxia telangiectasia mutated protein (ATM) and Artemis, the protein defective in patients with RS-SCID, function in a common double-strand break (DSB) repair pathway that also requires H2AX, 53BP1, Nbs1, Mre11, and DNA-PK. We show that radiation-induced Artemis hyperphosphorylation is ATM dependent. The DSB repair process requires Artemis nuclease activity and rejoins approximately 10% of radiation-induced DSBs. Our findings are consistent with a model in which ATM is required for Artemis-dependent processing of double-stranded ends with damaged termini. We demonstrate that Artemis is a downstream component of the ATM signaling pathway required uniquely for the DSB repair function but dispensable for ATM-dependent cell cycle checkpoint arrest. The significant radiosensitivity of Artemis-deficient cells demonstrates the importance of this component of DSB repair to survival.