Prevention of radiotherapy-induced side effects in retinoblastoma animal models

Retinoblastoma is the most common intraocular malignancy of infancy which is thought to originate from progenitor cells of the developing retina. The modern therapeutic approaches for retinoblastoma treatment including radiotherapy have very high success rates if the tumour is diagnosed at an early time point. But these therapies, especially radiotherapy, have side effects such as damage to healthy tissues and increasing the risk of induction of secondary tumours. As the radiotherapy has to be performed in a very young age and the risk of the therapy's side effects persist over lifetime, the quality of the patients' lives is strongly impaired. The risk of developing secondary tumours after radiotherapy is particularly high for patients with a family history of retinoblastoma, which leads to a deterioration in the quality of these patients’ lives. In this context, the aim of our project was to analyse a new option for the prevention of side effects of radiotherapy of the retinoblastoma through the use of the radioprotector ortho-phospho-L-tyrosine (pTyr). Three main questions had to be answered: 1. Is there a relevant retinoblastoma-mouse-model which can be used for radiotherapy? 2. Can the application of pTyr protect tissues that are damaged by irradiation during radiotherapy and prevent the induction of secondary tumours? 3. Does the application of pTyr interfere with the radiotherapy? To answer question 1., the retinoblastoma mouse models described in the literature were analysed. Since none of them fully met our requirements, we decided to establish a new retinoblastoma-xenograft-mouse model by performing intravitreal injections of human Y79 cells in immunodeficient nude mice. The growth of the xenograft tumours in the eyes was monitored in vivo by scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT). These results were compared to the results of the corresponding histological analysis. The in vivo and histological results showed a good correlation. The xenograft-tumours in the mouse-model showed very similar growth, histological and ultrastructural characteristics to Rb patients’ tumours and therefore our model can be used for the study of retinoblastoma. To answer the second question, Rb+/- mice as a model for patients who have defects in the homologue human RB1 gene were used. These patients have a higher risk of developing a retinoblastoma and other tumours like sarcomas, additionally, they are prone to radiotherapy-induced tumours. The animals were irradiated in a retinoblastoma radiotherapy-like procedure with and without a pTyr pre-treatment (100 mg/kg body weight, 16 hours before irradiation). A bleaching of the hair coat in the irradiated area started five weeks after irradiation in the animals which were not treated with pTyr. However, in the pTyr pre-treated animals the bleaching started later, about eight weeks after irradiation, and was significantly weaker. Importantly our in vivo (OCT) as well as our histological results have shown that pTyr protects the retina, which is a radiosensitive tissue, against the radiation-induced damage: the irradiation induced degenerative changes in the retina, such as thickness reduction, and loss of photoreceptors were significantly higher in the non-pTyr pretreated animals as compared to the pTyr-treated ones three, six and nine months after irradiation. No induced tumours or distant metastasis were found for any of the analysed mice. Since it is known that in retinoblastoma patients induced tumours are mostly diagnosed some 30 years after radiotherapy, the maximum lifespan of 16 months of our mice might be too short for the side effects to appear. Finally, in order to check if the application of pTyr would interfere with radiotherapy, the ability of pTyr to protect Y79 cells in vitro and in vivo was tested. For the in vitro testing Y 79 cell cultures were irradiated with and without pTyr pretreatment and their survival was analysed using different “limited dilution assays”. No significant radioprotection of pTyr on Y79 cells was detected. For the in vivo analysis of the radioprotection of Y79 cells by pTyr, our new established nude mouse model was used for a radiotherapeutic experiment with and without pTyr pre-treatment. The radiotherapy was successful as 80% of the mice which were not pre-treated with pTyr, stayed tumour-free for at least nine months after radiotherapy. However, in all pTyr pretreated animals a retinoblastoma reappeared. It has become apparent, that pTyr is a potent radioprotector for Y79- xenografts in contrast to Y79 cell cultures. This makes pTyr contraindicated for the radiotherapy of retinoblastoma. However, the radioprotective abilities of pTyr might make it interesting for the radiotherapy of other cancers, especially those with p53 mutations

Electromagnetic Biostimulation of Living Cultures for Biotechnology, Biofuel and Bioenergy Applications

The surge of interest in bioenergy has been marked with increasing efforts in research and development to identify new sources of biomass and to incorporate cutting-edge biotechnology to improve efficiency and increase yields. It is evident that various microorganisms will play an integral role in the development of this newly emerging industry, such as yeast for ethanol and Escherichia coli for fine chemical fermentation. However, it appears that microalgae have become the most promising prospect for biomass production due to their ability to grow fast, produce large quantities of lipids, carbohydrates and proteins, thrive in poor quality waters, sequester and recycle carbon dioxide from industrial flue gases and remove pollutants from industrial, agricultural and municipal wastewaters. In an attempt to better understand and manipulate microorganisms for optimum production capacity, many researchers have investigated alternative methods for stimulating their growth and metabolic behavior. One such novel approach is the use of electromagnetic fields for the stimulation of growth and metabolic cascades and controlling biochemical pathways. An effort has been made in this review to consolidate the information on the current status of biostimulation research to enhance microbial growth and metabolism using electromagnetic fields. It summarizes information on the biostimulatory effects on growth and other biological processes to obtain insight regarding factors and dosages that lead to the stimulation and also what kind of processes have been reportedly affected. Diverse mechanistic theories and explanations for biological effects of electromagnetic fields on intra and extracellular environment have been discussed. The foundations of biophysical interactions such as bioelectromagnetic and biophotonic communication and organization within living systems are expounded with special consideration for spatiotemporal aspects of electromagnetic topology, leading to the potential of multipolar electromagnetic systems. The future direction for the use of biostimulation using bioelectromagnetic, biophotonic and electrochemical methods have been proposed for biotechnology industries in general with emphasis on an holistic biofuel system encompassing production of algal biomass, its processing and conversion to biofuel

Data showing the shapes of cones and Müller cells within the fovea of monkeys reconstructed from serial sections and focused ion beam analysis

The data presented in this article are related to the research paper entitled “The anatomy of the foveola reinvestigated” (Tschulakow et al., 2018) [1]. Here we show the original aligned serial sections through the foveal centre of monkeys at different planes of section and 3 D models of central foveal cells. Keywords: Müller glial cells, Fovea, Cone receptors, Foveola, 3D model, Umbo, Stiles-Crawford effec

The anatomy of the foveola reinvestigated

Objective In the foveola of the eye, photoreceptors and Müller cells with a unique morphology have been described, but little is known about their 3D structure and orientation. Considering that there is an angle-dependent change in the foveolar photoreceptor response for the same light beam, known as the Stiles Crawford Effect of the first kind (SCE I), which is still not fully understood, a detailed analysis of the anatomy of the foveolar cells might help to clarify this phenomenon. Methods Serial semithin and ultrathin sections, and focused ion beam (FIB) tomography were prepared from 32 foveolae from monkeys (Macaca fascicularis) and humans. Foveolae were also analyzed under the electron microscope. Serial sections and FIB analysis were then used to construct 3D models of central Müller and photoreceptor cells. In addition, we measured the transmission of collimated light under the light microscope at different angles after it had passed through human foveae from flat mounted isolated retinae. Results In monkeys, outer segments of central foveolar cones are twice as long as those from parafoveal cones and do not run completely parallel to the incident light. Unique Müller cells are present in the central foveolae (area of 200 µm in diameter) of humans and monkeys. Light entering the fovea center, which is composed only of cones and Müller cells, at an angle of 0° causes a very bright spot after passing through this area. However, when the angle of the light beam is changed to 10°, less light is measured after transpasssing through the retina, the foveolar center becomes darker and the SCE-like phenomenon is directly visible. Measurements of the intensities of light transmission through the central foveola for the incident angles 0 and 10° resemble the relative luminance efficiency for narrow light bundles as a function of the location where the beam enters the pupil as reported by Stiles and Crawford. The effect persisted after carefully brushing away the outer segments. Conclusion We show that unique cones and Müller cells with light fibre-like properties are present in the center of the fovea. These unique Müller cells cause an angle dependent, SCE-like drop in the intensity of light guided through the foveola. Outer segments from the foveolar cones of monkeys are not straight

Establishment of a novel retinoblastoma (Rb) nude mouse model by intravitreal injection of human Rb Y79 cells – comparison of in vivo analysis versus histological follow up

Retinoblastoma (Rb) is the most frequent primary intraocular tumour in children and, if left untreated, can cause death. Preclinical animal models that mimic molecular, genetic, and cellular features of cancers are essential for studying cancer and searching for promising diagnosis and treatment modalities. There are several models described for Rb, but none of them fully meet our requirements. The aim of this study was to create a novel xenograft-nude mouse-model with broad application possibilities, which closely resembles the clinical observations of Rb patients and which could be used to investigate the development and spread of the tumour by using scanning laser ophthalmoscopy/optical coherence tomography (SLO/OCT) as well as histology methods. We injected human retinoblastoma Y79 cells intravitreally in both eyes of immune-deficient nude mice. The incidences of retinoblastoma as well as growth velocity were analysed 3, 6, 9 and 12 weeks after cell injection in vivo by SLO/OCT as well as ex vivo by electron microscopy (EM) and hematoxylin/eosin (HE) staining. Moreover, internal organs were histologically screened for potentially occurring metastases. Three weeks post-injection, animals developed a retinoblastoma, and after five weeks tumour growth resulted in swelling of the eyes in individual animals, showing a similar phenotype to that of untreated Rb patients at advanced stages of tumour-development. After 12 weeks, 67.5% of all analysed eyes (29 of 42) contained a retinoblastoma. At early stages of Rb development, the SLO/OCT analysis correlated with the histology results. If the tumours were too large, only histological investigations were feasible. The ultrastructural characteristics of the xenograft-tumours were very similar to those described for patient's tumours. In one mouse, brain metastases were observed. Our retinoblastoma mouse model closely resembles the human disease. SLO/OCT can be used for the detection of Rb at early stages of development and could be used for monitoring the success of future therapies

First study of oral artenimol-R in advanced cervical cancer: clinical benefit, tolerability and tumor markers

peer reviewedBackground/Aim: Artenimol-R is cytotoxic in transformed cervical cells and safety in humans is yet to be established. The present study investigates the clinical benefits, safety and the tumor marker effect of orally administered Artenimol-R in patients with advanced cervix carcinoma. Patients and Methods: Ten patients were treated with Artenimol-R for 28 days. Clinical symptoms, vaginal discharge and pain were followed-up. Adverse events were recorded. Biopsy samples were analyzed by immunohistochemistry for the expression of relevant tumor markers. Results: Artenimol-R treatment induced clinical remission with a median time for the disappearance of the symptoms being 7 days. No adverse events of grade 3 or 4 occurred. The expression of p53, Epidermal growth factor receptor (EGFR), and antigen Ki-67 as a cellular marker of proliferation, as well as the number of blood vessels stained by the CD31 antibody decreased, whereas the expression of transferrin receptor protein 1 (CD71) increased. Conclusion: The current pilot study provides evidence on the improvement of the clinical symptoms and the good tolerability of Artenimol-R in patients with advanced carcinoma of the cervix uteri. A survival trial with Artenimol- R in advanced patients is warranted