Molecular microbial ecology manual

The field of microbial ecology has been revolutionized in the past two decades by the introduction of molecular methods into the toolbox of the microbial ecologist. This molecular arsenal has helped to unveil the enormity of microbial diversity across the breadth of the earth's ecosystems, and has revealed that we are only familiar with a very small minority of the organisms that carry out key microbial functions in diverse habitats. The Molecular Microbial Ecology Manual, Second Edition (MMEM-II) provides a detailed and user-friendly description of the methods that have made this revolution in microbial ecology possible. However, what is perhaps most exciting about MMEM-II is that it contains a large number of new chapters, highlighting the newest trends in microbial ecology research, which seek to provide more quantitative and statistically robust data, and means of coupling microbial identity and function. In addition, the majority of the proven methods described in MMEM's first version have undergone significant revisions to provide the most up-to-date applications available. The state-of-the-art methods described in MMEM-II have not only been provided by experts in the field, but in most cases by the laboratories that actually first developed and applied the methods, thus providing the MMEM-II user with unique first-hand tips and insight

Immunophenotypical and functional heterogeneity of dendritic cells generated from murine bone marrow cultured with different cytokine combinations: implications for anti-tumoral cell therapy

Dendritic cells (DC) are professional antigen-presenting cells that can be used as immune adjuvant for anti-tumoural therapies. This approach requires the generation of large quantities of DC that are fully characterized on the immunophenotypical and functional levels. In a murine model, we analysed the in vitro effects of granulocyte–macrophage colony-stimulating factor (GM-CSF) alone or combined with interleukin-4 (IL-4) or Flt3 ligand (Flt3-L) on the number, immunophenotype and functions of bone marrow-derived DC. In GM-CSF cultures, we have identified two populations based on their level of expression of major histocompatibility complex (MHC) class II molecules: MHC-IIhi cells, exhibiting the typical morphology and immunophenotype of myeloid DC (CD11c+ 33D1+ DEC-205+ F4/80+), and MHC-IIlo cells, heterogeneous for DC markers (30% CD11c+; 50% 33D1+; DEC-205−; F4/80+). The addition of Flt3-L to GM-CSF induced a twofold increase in MHC-IIhi DC number; besides, the MHC-IIlo cells lost all DC markers. In contrast, after addition of IL-4 to GM-CSF, the two populations displayed a very similar phenotype (CD11c+ 33D1− DEC-205+ F4/80−), differing only in their expression levels of MHC class II and costimulatory molecules, and showed similar stimulatory activity in mixed leucocyte reaction. We next analysed the migration of these cultured cells after fluorescent labelling. Twenty-four hours after injection into the footpads of mice, fluorescent cells were detected in the draining popliteal lymph nodes, with an enhanced migration when cells were cultured with GM-CSF+Flt3-L. Finally, we showed that MHC-IIhi were more efficient than MHC-IIlo cells in an anti-tumoral vaccination protocol. Altogether, our data highlight the importance of characterizing in vitro-generated DC before use in immunotherapy