Direct Observation of Phagocytosis and NET-formation by Neutrophils in Infected Lungs using 2-photon Microscopy

After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity

Rapid Immunomagnetic Negative Enrichment of Neutrophil Granulocytes from Murine Bone Marrow for Functional Studies In Vitro and In Vivo

Polymorphonuclear neutrophils (PMN) mediate early immunity to infection but can also cause host damage if their effector functions are not controlled. Their lack or dysfunction is associated with severe health problems and thus the analysis of PMN physiology is a central issue. One prerequisite for PMN analysis is the availability of purified cells from primary organs. While human PMN are easily isolated from peripheral blood, this approach is less suitable for mice due to limited availability of blood. Instead, bone marrow (BM) is an easily available reservoir of murine PMN, but methods to obtain pure cells from BM are limited. We have developed a novel protocol allowing the isolation of highly pure untouched PMN from murine BM by negative immunomagnetic isolation using a complex antibody cocktail. The protocol is simple and fast (∼1 h), has a high yield (5–10*106 PMN per animal) and provides a purity of cells equivalent to positive selection (>80%). Most importantly, cells obtained by this method are non-activated and remain fully functional in vitro or after adoptive transfer into recipient animals. This method should thus greatly facilitate the study of primary murine PMN in vitro and in vivo

Current diagnostics and treatment of fibrosarcoma -perspectives for future therapeutic targets and strategies

Adult-type fibrosarcoma is a rare and highly aggressive subtype of soft tissue sarcomas. Due to the existence of other spindle-cell shaped sarcomas, its diagnosis is always one of exclusion. The likelihood of misdiagnoses between similar tumour entities is high, and often leads to inappropriate tumour treatment. We summarize here the main features of fibrosarcoma. When fibrosarcoma is appropriately diagnosed, the patient's overall prognosis is generally quite poor. Fibrosarcoma is characterized by its low sensitivity towards radio-and chemotherapy as well as by its high rate of tumour recurrences. Thus it is important to identify new methods to improve treatment of this tumour entity. We discuss some promising new directions in fibrosarcoma research, specifically focusing on more effective targeting of the tumour microenvironment. Communication between tumour cells and their surrounding stromal tissue play a crucial role in cancer progression, invasion, metastasis and chemosensitivity. The therapeutic potential of targeting the tumour microenvironment is addressed