Nephronophthisis - cause of chronic kidney disease in childhood. case report

Хроничното бъбречно заболяване (ХБЗ) в педиатричната популация се увеличава. Ранното откриване и лечение могат да намалят прогресията на бъбречните заболявания и да помогнат за превенцията на хронифицирането им. Дял за хронифициране на ХБЗ са наследствените заболявания.Нефронофтизата представлява наследствено бъбречно заболяване, характеризиращо се с хроничен тубуло-интерстициален нефрит, който може да се прояви в ранна детска възраст и в зависимост от формата да прогресира до терминална хронична бъбречна недостатъчност (ХБН).Водещи клинични прояви са: полиурия, полидипсия, анемия. Представяме клиничен случай на дете на 3 г. 9 м., често боледуващо от инфекции на горни дихателни пътища, станали повод за хоспитализация. От изследванията: Hb 80 g/l, урея 12 mmol/l, креатинин 156 μmol/l, урина - лекостепенна протеинурия, относително тегло 1010.От ултразвуковото изследване: двустранно бъбреци, гранични размери, повишена ехогенност на паренхима, данни за хипоехогенни зони в медулата, наподобяващи кистички. От направената биопсия: данни за атрофия на бъбречните тубули, с уплътняване на места на бъбречния интерстициум и единични кисти в медулата.Въз основа на клиничните прояви и резултатите от изследванията е поставена диагноза: нефронофтиза на Фанкони. Предвид малката възраст на детето и наличието на начален стадий на ХБЗ се налага адекватно проследяване и лечение с цел намаляване прогресията на ХБЗ.Chronic kidney disease (CKD) in the pediatric population increases. Early detection and treatment can reduce the progression of kidney diseases (KD) and help with the prevention of their chronification. Among the reasons for chronification of KD are hereditary diseases.Nephronophthisis is a hereditary kidney disease characterized by chronic tubulointerstitial nephritis which can manifest in infancy and depending on the type - progress to end-stage CKD. Leading clinical manifestations are: polyuria, polydipsia, anemia.We present a case of a child of 3 years. 9m., often suffering from infections of the upper respiratory tract. One of those infections became a reason for hospitalization. From laboratory tests: Hb 80 g/l, urea 12 mmol/l, creatinine 156 μmol/l, urine - mild proteinuria, density 1010.Ultrasonography results: bilateral kidneys, border sized, increased renal parenchymal echogenicity, data for hypoechoic areas in the medulla resembling small cysts.Biopsy results: evidence of atrophy of the renal tubules, increased incidence of interstitial cells in the renal interstitium, single cysts in the medulla. Based on the clinical manifestations and the results of lab tests the patient is diagnosed with nephronophthisis (NPH1). Given the child`s young age and the presence of early stages of CKD sufficient monitor ing and treatment are needed to reduce the progression of CKD

Assessment of drug-loaded nanoparticles in a 3D in vitro brain tumour model

This work describes the creation of a three-dimensional model of the children’s brain tumour medulloblastoma using primarily human cells. This in vitro cell culture model was created as a platform for testing novel drug delivery systems for local delivery in the brain. The aim of the local delivery strategy was to reduce radiotherapy through the use of nanoparticle-based chemotherapy. The nanoparticles would be delivered after surgery in the cavity left by the excised tumour tissue. The model was intended to evaluate the selective cytotoxicity of advanced drug delivery systems towards tumour tissue and the benefit of nanoparticle therapy compared to free drug. Normal tissue was modelled using human foetal brain tissue and tumour tissue was represented by a variety of medulloblastoma cell lines. Both were cultured as three-dimensional spheroids free of artificial matrix in ultra-low attachment plates. The tumour and normal cells could be cultured either separately or together and the viability for each cell population determined using a battery of methods. Co-cultures of both cell types had the additional benefit of mimicking the interaction between normal and tumour tissue. The use of physiologically relevant single and co-culture in vitro models could provide information on the relative safety and efficacy of novel brain tumour treatments. The high-throughput platforms used, the algorithms and the validation of a battery of tests in 3D may be extrapolated to other cancer models as well. Moreover the universal marking procedure employed can be employed to label, culture and analyse any two cell types, while preserving tissue heterogeneity and viability. The key benefit from this thesis is the framework for designing in vitro models of tumours that include normal tissue as an internal control. This is an important contribution that can substantiate IC50 values by putting them in the context of drug safety and efficacy. It also highlights the minimum checks and feasibility experiments that need to be done before an in vitro assay is accepted for 3D spheroids

Assessment of drug-loaded nanoparticles in a 3D in vitro brain tumour model

This work describes the creation of a three-dimensional model of the children’s brain tumour medulloblastoma using primarily human cells. This in vitro cell culture model was created as a platform for testing novel drug delivery systems for local delivery in the brain. The aim of the local delivery strategy was to reduce radiotherapy through the use of nanoparticle-based chemotherapy. The nanoparticles would be delivered after surgery in the cavity left by the excised tumour tissue. The model was intended to evaluate the selective cytotoxicity of advanced drug delivery systems towards tumour tissue and the benefit of nanoparticle therapy compared to free drug. Normal tissue was modelled using human foetal brain tissue and tumour tissue was represented by a variety of medulloblastoma cell lines. Both were cultured as three-dimensional spheroids free of artificial matrix in ultra-low attachment plates. The tumour and normal cells could be cultured either separately or together and the viability for each cell population determined using a battery of methods. Co-cultures of both cell types had the additional benefit of mimicking the interaction between normal and tumour tissue. The use of physiologically relevant single and co-culture in vitro models could provide information on the relative safety and efficacy of novel brain tumour treatments. The high-throughput platforms used, the algorithms and the validation of a battery of tests in 3D may be extrapolated to other cancer models as well. Moreover the universal marking procedure employed can be employed to label, culture and analyse any two cell types, while preserving tissue heterogeneity and viability. The key benefit from this thesis is the framework for designing in vitro models of tumours that include normal tissue as an internal control. This is an important contribution that can substantiate IC50 values by putting them in the context of drug safety and efficacy. It also highlights the minimum checks and feasibility experiments that need to be done before an in vitro assay is accepted for 3D spheroids

NАТ technology – a step forward in hepatitis C screening in Bulgaria

Introduction: Globally, blood transfusions are the cause of transmissible infections in between 10% and 15% of all patients. Currently, the testing of donor blood for transmissible infections is utilizing enzyme-linked immunosorbent assay ELISA and chemiluminescence method CLIA for screening and detection of hepatitis C (HCV Ag/Ab). Since February, 2020 all donor samples of blood taken in the 5 centers for transfusion hematology in Bulgaria have been tested for markers of transmissible infections, including HCV, by nucleic acid testing (NAT).Aim: We have set the goal to analyze data produced by the implementation of NAT testing of donor blood for the period of 4 months (from 10th of February to 16th of June, 2020) regarding HCV infected samples.Materials and Methods: We present our experience of using NAT and data produced by its implementation for testing of donor blood for a period of 4 months, from the time NAT technology was implemented in Bulgaria.Results and Discussion: The performance of the multiplex Procleix Ultrio Elite assay as individual donor nucleic acid test for the detection of HCV is evaluated in a retrospective study. An advantage of the method is that it not only allows for timely identification of infected donor blood and saves lives of the recipients but also saves the lives of donors, as treatment of chronic hepatitis C continues to progress and is now effective in clearing the virus in more than half of the patients. In terms of safety and security of diagnosis and blood products obtained, the method has no alternative.Conclusion: A recommendation is drafted: to adopt a strategy to increase the quality of healthcare by introducing a one-time HCV screening for adults aged 18 and above, for individuals at risk and for pregnant women to reduce further the risk of incidental transmission of the hepatitis C virus

Contribution of sulfurous mineral waters to dietary sulfur intake and metabolism

Sulfur is a non-metal macroelement with critical importance for the human body integrity and homeostasis. Sulfur-containing biomolecules exert important functions in redox balance maintenance, enzyme functionality, DNA methylation and repair, modification of extracellular matrix components, and xenobiotic metabolism.Many studies related to the sulfur utilization and metabolism are focused on foods rich in organosulfur compounds that are associated with health benefits. It is believed that sulfur-containing mineral water also could have beneficial effects on the human health, but this knowledge is currently based on empirical data.It could be suggested that the intake of sulfurous mineral waters as a part of the everyday diet would have measurable effects on the human metabolism

Multiplexing Spheroid Volume, Resazurin and Acid Phosphatase Viability Assays for High-Throughput Screening of Tumour Spheroids and Stem Cell Neurospheres

Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money

Separating chemotherapy-related developmental neurotoxicity from cytotoxicity in monolayer and neurosphere cultures of human fetal brain cells

Chemotherapy-induced neurotoxicity can reduce the quality of life of patients by affecting their intelligence, senses and mobility. Ten percent of safety-related late-stage clinical failures are due to neurological side effects. Animal models are poor in predicting human neurotoxicity due to interspecies differences and most in vitro assays cannot distinguish neurotoxicity from general cytotoxicity for chemotherapeutics. We developed in vitro assays capable of quantifying the paediatric neurotoxic potential for cytotoxic drugs. Mixed cultures of human fetal brain cells were differentiated in monolayers and as 3D-neurospheres in the presence of non-neurotoxic chemotherapeutics (etoposide, teniposide) or neurotoxicants (methylmercury). The cytotoxic potency towards dividing progenitors versus differentiated neurons and astrocytes was compared using: (1) immunohistochemistry staining and cell counts in monolayers; (2) through quantitative Western blots in neurospheres; and (3) neurosphere migration assays. Etoposide and teniposide, were 5–10 times less toxic to differentiated neurons compared to the mix of all cells in monolayer cultures. In contrast, the neurotoxicant methylmercury did not exhibit selectivity and killed all cells with the same potency. In 3D neurospheres, etoposide and teniposide were 24 to 10 times less active against neurons compared to all cells. These assays can be used prioritise drugs for local drug delivery to brain tumours

Highlights of children with Cancer UK’s workshop on drug delivery in paediatric brain tumours

The first Workshop on Drug Delivery in Paediatric Brain Tumours was hosted in London by the charity Children with Cancer UK. The goals of the workshop were to break down the barriers to treating central nervous system (CNS) tumours in children, leading to new collaborations and further innovations in this under-represented and emotive field. These barriers include the physical delivery challenges presented by the blood–brain barrier, the underpinning reasons for the intractability of CNS cancers, and the practical difficulties of delivering cancer treatment to the brains of children. Novel techniques for overcoming these problems were discussed, new models brought forth, and experiences compared

Synthesis and properties of a biodegradable polymer-drug conjugate: Methotrexate-poly(glycerol adipate)

Polymer-drug conjugates have been actively developed as potential anticancer drug delivery systems. In this study, we report the first polymer-anticancer drug conjugate with poly(glycerol adipate) (PGA) through the successful conjugation of methotrexate (MTX). MTX-PGA conjugates were controllably and simply fabricated by carbodiimide-mediated coupling reaction with various high molar ratios of MTX. The MTX-PGA conjugate self-assembled into nanoparticles with size dependent on the amount of conjugated MTX and the pH of medium. Change in particle size was attributed to steric hindrance and bulkiness inside the nanoparticle core and dissociation of free functional groups of the drug. The MTX-PGA nanoparticles were physically stable in media with pH range of 5–9 and ionic strength of up to 0.15 M NaCl and further chemically stable against hydrolysis in pH 7.4 medium over 30 days but enzymatically degradable to release unchanged free drug. Although 30%MTX-PGA nanoparticles exhibited only slightly less potency than free MTX in 791T cells in contrast to previously reported human serum albumin-MTX conjugates which had >300 times lower potency than free MTX. However, the MTX nanoparticles showed 7 times higher toxicity to Saos-2 cells than MTX. Together with the enzymic degradation experiments, these results suggest that with a suitable biodegradable polymer a linker moiety is not a necessary component. These easily synthesised PGA drug conjugates lacking a linker moiety could therefore be an effective new pathway for development of polymer drug conjugates

In vitro models of medulloblastoma: choosing the right tool for the job

The recently-defined four molecular subgroups of medulloblastoma have required updating of our understanding of in vitro models to include molecular classification and risk stratification features from clinical practice. This review seeks to build a more comprehensive picture of the in vitro systems available for modelling medulloblastoma. The subtype classification and molecular characterisation for over 40 medulloblastoma cell-lines has been compiled, making it possible to identify the strengths and weaknesses in current model systems. Less than half (18/44) of established medulloblastoma cell-lines have been subgrouped. The majority of the subgrouped cell-lines (11/18) are Group 3 with MYC-amplification. SHH cell-lines are the next most common (4/18), half of which exhibit TP53 mutation. WNT and Group 4 subgroups, accounting for 50% of patients, remain underrepresented with 1 and 2 cell-lines respectively. In vitro modelling relies not only on incorporating appropriate tumour cells, but also on using systems with the relevant tissue architecture and phenotype as well as normal tissues. Novel ways of improving the clinical relevance of in vitro models are reviewed, focusing on 3D cell culture, extracellular matrix, co-cultures with normal cells and organotypic slices. This paper champions the establishment of a collaborative online-database and linked cell-bank to catalyse preclinical medulloblastoma research