Direct measurement of the 3-dimensional DNA lesion distribution induced by energetic charged particles in a mouse model tissue.

Charged particles are increasingly used in cancer radiotherapy and contribute significantly to the natural radiation risk. The difference in the biological effects of high-energy charged particles compared with X-rays or γ-rays is determined largely by the spatial distribution of their energy deposition events. Part of the energy is deposited in a densely ionizing manner in the inner part of the track, with the remainder spread out more sparsely over the outer track region. Our knowledge about the dose distribution is derived solely from modeling approaches and physical measurements in inorganic material. Here we exploited the exceptional sensitivity of γH2AX foci technology and quantified the spatial distribution of DNA lesions induced by charged particles in a mouse model tissue. We observed that charged particles damage tissue nonhomogenously, with single cells receiving high doses and many other cells exposed to isolated damage resulting from high-energy secondary electrons. Using calibration experiments, we transformed the 3D lesion distribution into a dose distribution and compared it with predictions from modeling approaches. We obtained a radial dose distribution with sub-micrometer resolution that decreased with increasing distance to the particle path following a 1/r2 dependency. The analysis further revealed the existence of a background dose at larger distances from the particle path arising from overlapping dose deposition events from independent particles. Our study provides, to our knowledge, the first quantification of the spatial dose distribution of charged particles in biologically relevant material, and will serve as a benchmark for biophysical models that predict the biological effects of these particles.

Refinement of water-filtered infrared A (wIRA) irradiations of in vitro acute and persistent chlamydial infections.

Water-filtered infrared A (wIRA) alone or in combination with visible light (VIS) exerts anti-chlamydial effects in vitro and in vivo in acute infection models. However, it has remained unclear whether reduced irradiation duration and irradiance would still maintain anti-chlamydial efficacy. Furthermore, efficacy of this non-chemical treatment option against persistent (chronic) chlamydial infections has not been investigated to date. To address this knowledge gap, we evaluated 1) irradiation durations of 5, 15 or 30 min in genital and ocular Chlamydia trachomatis acute infection models, 2) irradiances of 100, 150 or 200 mW/cm2 in the acute genital infection model and 3) anti-chlamydial activity of wIRA and VIS against C. trachomatis serovar B and E with amoxicillin (AMX)- or interferon γ (IFN-γ)-induced persistence. Reduction of irradiation duration reduced anti-chlamydial efficacy. Irradiances of 150 to 200 mW/cm2, but not 100 mW/cm2, induced anti-chlamydial effects. For persistent infections, wIRA and VIS irradiation showed robust anti-chlamydial activity independent of the infection status (persistent or recovering), persistence inducer (AMX or IFN-γ) or chlamydial strain (serovar B or E). This study clarifies the requirement of 30 min irradiation duration and 150 mW/cm2 irradiance to induce significant anti-chlamydial effects in vitro, supports the use of irradiation in the wIRA and VIS spectrum as a promising non-chemical treatment for chlamydial infections and provides important information for follow-up in vivo studies. Notably, wIRA and VIS exert anti-chlamydial effects on persistent chlamydiae which are known to be refractory to antibiotic treatment.

Effects of water-filtered infrared A and visible light (wIRA/VIS) radiation on heat- and stress-responsive proteins in the retina and cornea of guinea pigs.

Water-filtered infrared A and visible light (wIRA/VIS), shown to reduce chlamydial infections in vitro and in vivo, might represent an innovative therapeutic approach against trachoma, a neglected tropical disease caused by ocular infection with the bacterium C. trachomatis. In this in vivo study, we assessed the impact of wIRA radiation in combination with VIS (wavelength range 595-1400 nm, intensity 2100 W/m2) on the retina and cornea in a guinea pig animal model of inclusion conjunctivitis. We investigated the effects 19 days after wIRA/VIS irradiation by comparing a single and double wIRA/VIS treatment with a sham control. By immunolabeling and western blot analyses of critical heat- and stress-responsive proteins, we could not detect wIRA/VIS-induced changes in their expression pattern. Also, immunolabeling of specific retinal marker proteins revealed no changes in their expression pattern caused by the treatment. Our preclinical study suggests wIRA/VIS as a promising and safe therapeutic tool to treat ocular chlamydial infections.

Differences in the Response to DNA Double-Strand Breaks between Rod Photoreceptors of Rodents, Pigs, and Humans.

Genome editing (GE) represents a powerful approach to fight inherited blinding diseases in which the underlying mutations cause the degeneration of the light sensing photoreceptor cells of the retina. Successful GE requires the efficient repair of DNA double-stranded breaks (DSBs) generated during the treatment. Rod photoreceptors of adult mice have a highly specialized chromatin organization, do not efficiently express a variety of DSB response genes and repair DSBs very inefficiently. The DSB repair efficiency in rods of other species including humans is unknown. Here, we used ionizing radiation to analyze the DSB response in rods of various nocturnal and diurnal species, including genetically modified mice, pigs, and humans. We show that the inefficient repair of DSBs in adult mouse rods does not result from their specialized chromatin organization. Instead, the DSB repair efficiency in rods correlates with the level of Kruppel-associated protein-1 (KAP1) expression and its ataxia-telangiectasia mutated (ATM)-dependent phosphorylation. Strikingly, we detected robust KAP1 expression and phosphorylation only in human rods but not in rods of other diurnal species including pigs. Hence, our study provides important information about the uniqueness of the DSB response in human rods which needs to be considered when choosing model systems for the development of GE strategies.

Water-filtered Infrared A and visible light (wIRA/VIS) treatment reduces Chlamydia caviae-induced ocular inflammation and infectious load in a Guinea pig model of inclusion conjunctivitis.

Trachoma is a devastating neglected tropical disease caused by Chlamydia trachomatis and the leading global cause of infectious blindness. Although antibiotic treatment against trachoma is efficient (SAFE strategy), additional affordable therapeutic strategies are of high interest. Water-filtered infrared A and visible light (wIRA/VIS) irradiation has proven to reduce chlamydial infectivity in vitro and ex vivo. The aim of this study was to evaluate whether wIRA/VIS can reduce chlamydial infection load and/or ocular pathology in vivo, in a guinea pig model of inclusion conjunctivitis. Guinea pigs were infected with 1 × 106 inclusion-forming units/eye of Chlamydia caviae via the ocular conjunctiva on day 0. In infected animals, wIRA/VIS irradiation (2100 W/m2) was applied on day 2 (single treatment) and on days 2 and 4 (double treatment) post-infection (pi). wIRA/VIS reduced the clinical pathology score on days 7 and 14 pi and the conjunctival chlamydial load on days 2, 4, 7, and 14 pi in comparison with C. caviae-infected, not irradiated, controls. Furthermore, numbers of chlamydial inclusions were decreased in wIRA/VIS treated C. caviae-infected guinea pigs on day 21 pi compared to C. caviae-infected, non-irradiated, controls. Double treatment with wIRA/VIS (days 2 and 4 pi) was more efficient than a single treatment on day 2 pi. wIRA/VIS treatment did neither induce macroscopic nor histologic changes in ocular tissues. Our results indicate that wIRA/VIS shows promising efficacy to reduce chlamydial infectivity in vivo without causing irradiation related pathologies in the follow-up period. wIRA/VIS irradiation is a promising approach to reduce trachoma transmission and pathology of ocular chlamydial infection.

Water-filtered infrared A reduces chlamydial infectivity in vitro without causing ex vivo eye damage in pig and mouse models.

Repeated ocular infections with Chlamydia trachomatis trigger the development of trachoma, the most common cause of infectious blindness worldwide. Water-filtered infrared A (wIRA) has shown positive effects on cultured cells and human skin. Our aim was to evaluate the potential of wIRA as a possible non-chemical treatment for trachoma patients. We both modeled ocular chlamydial infections using C. trachomatis B to infect human conjunctival epithelial cells (HCjE) and studied the effects of wIRA on non-infected ocular structures with two ex vivo eye models. We focused on the temperature development during wIRA irradiation in cell culture and perfused pig eyes to exclude potentially harmful side effects. Furthermore, cell viability of HCjE and cytotoxicity in mouse retina explants was analyzed. We demonstrated a significant wIRA-dependent reduction of chlamydial infectivity in HCjE cells. Moreover, we observed that wIRA treatment of HCjE prior to infection was sufficient to inhibit chlamydial infectivity and that visible light enhances the effect of wIRA. Irradiation did not reduce cell viability and there was no indication of retinal damage post treatment. Additionally, temperatures during wIRA exposure did not markedly exceed physiological eye temperatures, suggesting that hyperthermia-related lesions are unlikely. For clinical applications, further exploration of wIRA as a non-chemical treatment device in an experimental animal model is essential.

Inefficient double-strand break repair in murine rod photoreceptors with inverted heterochromatin organization.

BACKGROUND: DNA double-strand break (DSB) repair is crucial for the maintenance of genomic stability, and chromatin organization represents one important factor influencing repair efficiency. Mouse rod photoreceptors with their inverted heterochromatin organization containing a single large chromocenter in the middle of the nucleus provide a unique model system to study DSB repair in heterochromatin of living animals. RESULTS: We observed that adult rod photoreceptors repair only half of the induced DSBs within 1 day after damage induction, a defect that is neither observed in any other cell type of the adult retina nor in rod photoreceptor precursor cells of postnatal day 4 mice. We show that adult wild-type rods are deficient in a repair pathway involving ATM, a protein that promotes heterochromatic DSB repair by phosphorylating KAP1 and facilitating heterochromatin relaxation. Of note, we observed that rods fail to robustly accumulate active ATM at DSBs, exhibit low KAP1 levels, and display high levels of SPOC1, a factor suppressing KAP1 phosphorylation. Collectively, this results in dramatically reduced KAP1 phosphorylation and the inability to repair heterochromatic DSBs. CONCLUSIONS: Because the distinct heterochromatic structure of rods focuses transmitting light to enable vision at low photon levels, the inability to phosphorylate KAP1 and the failure to relax heterochromatin could serve to maintain this structure and the functionality of rods in the presence of DSBs. Collectively, our findings show that the unique chromatin organization of adult rods renders them incapable to efficiently repair heterochromatic DSBs, providing evidence that heterochromatin affects mammalian DSB repair in vivo.

Near-infrared exposure changes cellular responses to ionizing radiation.

Near infrared (NIR) and X-rays are radiations from different sides of the wavelength spectrum but both are used during medical treatments, as they have severe impacts on cellular processes, including metabolism, gene expression, proliferation and survival. However, both radiations differ strictly in their consequences for exposed patients: NIR effects are generally supposed to be positive, mostly ascribed to a stimulation of metabolism, whereas X-ray leads to genetic instability, an increase of reactive oxygen species (ROS) and DNA damages and finally to cellular death by apoptosis in tumor cells. Since genomic stability after X-irradiation depends on the mitochondrial metabolism, which is well known to be regulated by NIR, we analyzed the impact of NIR on cellular responses of fibroblasts, retinal progenitor cells and keratinocytes to X-radiation. Our data show that previous exposure to naturally occurring doses of nonthermal NIR combined with clinically relevant X-ray doses leads to (1) increased genomic instability, indicated by elevated ratios of mitotic catastrophes, (2) increased ROS, (3) higher amounts of X-irradiated cells entering S-phase and (4) impaired DNA double-strand break repair. Taken together, our data show tremendous effects of NIR on cellular responses to X-rays, probably affecting the results of radiotherapy after NIR exposure during cancer treatment.