High-throughput spheroid screens using volume, resazurin reduction and acid phosphatase activity

Mainstream adoption of physiologically-relevant three-dimensional models has been slow in the last 50 years due to long, manual protocols with poor reproducibility, high price and closed commercial platforms. This chapter describes high-throughput, low-cost, open methods for spheroid viability assessment which use readily-available reagents and open-source software to analyse spheroid volume, metabolism and enzymatic activity. We provide two ImageJ macros for automated spheroid size determination - for both single images and for images in stacks. We also share an Excel template spreadsheet allowing users to rapidly process spheroid size data, analyse plate uniformity (such as edge effects and systematic seeding errors), detect outliers and calculate dose-response. The methods would be useful to researchers in preclinical and translational research planning to move away from simplistic monolayer studies and explore 3D spheroid screens for drug safety and efficacy without substantial investment in money or time

CCAAT/Enhancer-Binding Protein γ Is a Critical Regulator of IL-1β-Induced IL-6 Production in Alveolar Epithelial Cells

CCAAT/enhancer binding protein γ (C/EBPγ) is a member of the C/EBP family of transcription factors, which lacks known activation domains. C/EBPγ was originally described as an inhibitor of C/EBP transactivation potential. However, previous study demonstrates that C/EBPγ augments the C/EBPβ stimulatory activity in lipopolysaccharide induction of IL-6 promoter in a B lymphoblast cell line. These data indicate a complexing functional role for C/EBPγ in regulating gene expression. Furthermore, the expression and function of C/EBPγ during inflammation are still largely unknown. In this study, we demonstrate that C/EBPγ activation was induced by IL-1β treatment in lung epithelial cells. Importantly, we demonstrate for the first time that C/EBPγ plays a critical role in regulating IL-1β-induced IL-6 expression in both mouse primary alveolar type II epithelial cells and a lung epithelial cell line, MLE12. We further provide the evidence that C/EBPγ inhibits IL-6 expression by inhibiting C/EBPβ but not NF-κB stimulatory activity in MLE12 cells. These findings suggest that C/EBPγ is a key transcription factor that regulates the IL-6 expression in alveolar epithelial cells, and may play an important regulatory role in lung inflammatory responses

Особенности преподавания фтизиатрии на примере разбора врачебных ошибок

In the educational program of the Chair of Tuberculosis and Pulmonology of Siberian State Medical University (Tomsk), there is a special consideration towards tuberculosis alarm, the ways of TB detection, diagnostics and differential diagnostics of pulmonary and extra-pulmonary TB. Moreover, there is an educational promotion of deontology and medical ethics questions to students and listeners of the Advance Training Faculty and the Post-graduate Professional Education Course. They also participate in review conferences related to causes and ways of prevention medical malpractice cases.На кафедре фтизиатрии и пульмонологии Сибирского государственного медицинского университета (г. Томск) в процессе обучения делается упор на фтизиатрическую настороженность, пути и способы выявления, диагностики и дифференциальной диагностики легочного и внелегочного туберкулеза. Такие вопросы, как деонтология, медицинская этика, раскрываются студентам и слушателям ФПК и ППС в процессе обучения, проводятся разборы причин и пути предотвращения врачебных ошибок

Результаты игловой биопсии плевры в диагностике экссудативных плевритов

Summary. In this study, diagnostic value of pleural needle biopsy has been estimated. Biopsy specimens were obtained from 226 patients with pleural exudates of different etiologies. A high diagnostic sensitivity of this procedure was estimated under the lowest rate of complications. Резюме. Целью исследования являлось определение диагностической ценности игловой биопсии плевры (ИБП) при плевральной патологии. С марта 2006 по январь 2011 г. было выполнено 226 ИБП. Возраст пациентов, среди которых мужчин было 160 (70,8 %), женщин – 66 (29,2 %), варьировался от 18–84 лет. В 39,8 % случаев (у 90 больных) биопсия проводилась по поводу изолированного плеврита, в 60,2 % (у 136) – при плеврите, осложнившем основной процесс в легком или другом экстраторакальном органе. Проведенное исследование показало, что ИБП является высокоинформативным методом диагностики, а его чувствительность при выявлении туберкулезного плеврита составляет 70,2 %

Screening out irrelevant cell-based models of disease

The common and persistent failures to translate promising preclinical drug candidates into clinical success highlight the limited effectiveness of disease models currently used in drug discovery. An apparent reluctance to explore and adopt alternative cell-and tissue-based model systems, coupled with a detachment from clinical practice during assay validation, contributes to ineffective translational research. To help address these issues and stimulate debate, here we propose a set of principles to facilitate the definition and development of disease-relevant assays, and we discuss new opportunities for exploiting the latest advances in cell-based assay technologies in drug discovery, including induced pluripotent stem cells, three-dimensional (3D) co-culture and organ-on-a-chip systems, complemented by advances in single-cell imaging and gene editing technologies. Funding to support precompetitive, multidisciplinary collaborations to develop novel preclinical models and cell-based screening technologies could have a key role in improving their clinical relevance, and ultimately increase clinical success rates

Novel three-dimensional (3D) microtissues for the discovery of chemoradiation-sensitizing compounds.

The rapid evolution of resistance to both conventional and small molecule therapies is a challenging problem in oncology. One approach to overcome resistance is to use combinatorial treatments that exploit the synergies between diff erent therapy modalities. The combination of chemotherapy and radiation treatment is emerging as a potentially effective combinatorial regimen, although the optimal mix has not been identified so far. 3D tumor spheroids have been used as relevant biologic al models for oncology drug discovery for many years, as 3D cell culture systems can provide a better representation of tissue - level biology over classical 2D culture systems. However, aside from chemotherapeutic interventions, these 3D models are also hig hly valuable tools for emulating radiotherapy in vitro . We have adopted innovative 3D - microtissue 96 - well plate technology for the screening of potential radio - sensitizing compounds on tumour cells. We followed the response to radiation and drug - treatment of radiation on breast cancer and glioblastoma cells stably transduced with a GFP - expressing lentiviral vector. High throughput quantification of tumour 3D - microtissue growth was assessed in real time using the high content imaging platform (PerkinElmer, U SA). We have validated the tumour 3D - microtissue model by comparing the treatment of microtissues with different chemotherapeutic compounds used in the clinic and additionally analyzed novel HDAC inhibitors and MEK inhibitors of the MAPK signalling pathwa y. Results for Docetaxel, Doxorubicine, 5 - FU, Vinblastine, SAHA and MEK1 inhibitor treatment showed that the corresponding IC50 values were improved by a single 2Gy or 4Gy radiation dose on radioresistant tumour cells. This screen confirms that the assay u sing the 3D - microtissue model system is able to detect novel compounds that modulate tumour cell survival after irradiation