Identifying Activated T Cells in Reconstituted RAG Deficient Mice Using Retrovirally Transduced Pax5 Deficient Pro-B Cells

Various methods have been used to identify activated T cells such as binding of MHC tetramers and expression of cell surface markers in addition to cytokine-based assays. In contrast to these published methods, we here describe a strategy to identify T cells that respond to any antigen and track the fate of these activated T cells. We constructed a retroviral double-reporter construct with enhanced green fluorescence protein (EGFP) and a far-red fluorescent protein from Heteractis crispa (HcRed). LTR-driven EGFP expression was used to enrich and identify transduced cells, while HcRed expression is driven by the CD40Ligand (CD40L) promoter, which is inducible and enables the identification and cell fate tracing of T cells that have responded to infection/inflammation. Pax5 deficient pro-B cells that can give rise to different hematopoietic cells like T cells, were retrovirally transduced with this double-reporter cassette and were used to reconstitute the T cell pool in RAG1 deifcient mice that lack T and B cells. By using flow cytometry and histology, we identified activated T cells that had developed from Pax5 deficient pro-B cells and responded to infection with the bacterial pathogen Listeria monocytogenes. Microscopic examination of organ sections allowed visual identification of HcRed-expressing cells. To further characterize the immune response to a given stimuli, this strategy can be easily adapted to identify other cells of the hematopoietic system that respond to infection/inflammation. This can be achieved by using an inducible reporter, choosing the appropriate promoter, and reconstituting mice lacking cells of interest by injecting gene-modified Pax5 deficient pro-B cells

WSES guidelines for management of Clostridium difficile infection in surgical patients

In the last two decades there have been dramatic changes in the epidemiology of Clostridium difficile infection (CDI), with increases in incidence and severity of disease in many countries worldwide. The incidence of CDI has also increased in surgical patients. Optimization of management of C difficile, has therefore become increasingly urgent. An international multidisciplinary panel of experts prepared evidenced-based World Society of Emergency Surgery (WSES) guidelines for management of CDI in surgical patients.Peer reviewe

The Small School: A Case Study of School Education

Krátce po zahájení reformy základního vzdělávání v České republice v roce 2007 jsme za použití strategie případové studie zkoumali vyučování, učení a pedagogické řízení v konkrétní základní škole. Pro popis vzdělávání ve škole jsme užívali kvalitativní i kvantitativní metody sběru dat – především dotazování, pozorování výuky a analýzu dokumentů. Za použití kvalitativních postupů analýzy dat jsme nastínili obraz zaměřující pozornost především na vývoj školy v posledních letech a na strukturní determinanty její práce, jako jsou velikost školy a koexistence primárního a nižšího sekundárního stupně. Výsledky studie zachycují obraz školního vzdělávání na počátku kurikulární reformy a současně slouží pro konkretizaci plánů následujících případových studií dalších škol.A major educational reform of the primary and lower secondary schools started in the Czech Republic in 2007. Shortly after the launch of the reform, we studied the actual picture of teaching, learning, and instructional leadership in one Czech comprehensive school, using the design research of a case study. Both qualitative and quantitative methods were used to describe the educational processes in the school – predominantly questioning, observation, and documentary analysis. Using qualitative data analysis we have sketched a narrative that highlights the role of structural factors– the small size of the school, the co-existence of primary and lower secondary schools within one organisation, as well as its unique history. The research results describe a case of schooling at the very beginning of educational reform, and guide the design of our next case studies of schools

Pax2/5 and Pax6 subdivide the early neural tube into three domains

The nested expression patterns of the paired−box containing transcription factors Pax2/5 and Pax6 demarcate the midbrain and forebrain primordium at the neural plate stage. We demonstrate that, in Pax2/5 deficient mice, the mesencephalon/metencephalon primordium is completely missing, resulting in a fusion of the forebrain to the hindbrain. Morphologically, in the alar plate the deletion is characterized by the substitution of the tectum (dorsal midbrain) and cerebellum (dorsal metencephalon) by the caudal diencephalon and in the basal plate by the replacement of the midbrain tegmentum by the ventral metencephalon (pons). Molecularly, the loss of the tectum is demonstrated by an expanded expression of Pax6, (the molecular determinant of posterior commissure), and a rostral shift of the territory of expression of Gbx2 and Otp (markers for the pons), towards the caudal diencephalon. Our results suggest that an intact territory of expression of Pax2/5 in the neural plate, nested between the rostral and caudal territories of expression of Pax6, is necessary for defining the midbrain vesicle. Ó 1999 Elsevier Science Ireland Ltd. All rights reserve