Early sensing of Yersinia pestis airway infection by bone marrow cells.

Bacterial infection of the lungs triggers a swift innate immune response that involves the production of cytokines and chemokines that promote recruitment of immune cells from the bone marrow (BM) into the infected tissue and limit the ability of the pathogen to replicate. Recent in vivo studies of pneumonic plague in animal models indicate that the pulmonary pro-inflammatory response to airway infection with Yersinia pestis is substantially delayed in comparison to other pathogens. Consequently, uncontrolled proliferation of the pathogen in the lungs is observed, followed by dissemination to internal organs and death. While the lack of an adequate early immune response in the lung is well described, the response of BM-derived cells is poorly understood. In this study, we show that intranasal (i.n.) infection of mice with a fully virulent Y. pestis strain is sensed early by the BM compartment, resulting in a reduction in CXCR4 levels on BM neutrophils and their subsequent release into the blood 12 hours (h) post infection. In addition, increased levels of BM-derived hematopoietic stem and progenitor cells (HSPC) were detected in the blood early after infection. Mobilization of both immature and mature cells was accompanied by the reduction of BM SDF-1 (CXCL-12) levels and the reciprocal elevation of SDF-1 in the blood 24 h post infection. RT-PCR analysis of RNA collected from total BM cells revealed an early induction of myeloid-associated genes, suggesting a prompt commitment to myeloid lineage differentiation. These findings indicate that lung infection by Y. pestis is sensed by BM cells early after infection, although bacterial colonization of the BM occurs at late disease stages, and point on a potential cross-talk between the lung and the BM at early stages of pneumonic plague.

Application of nanoparticles for the detection and sorting of pathogenic bacteria by flow-cytometry.

In this paper we will describe a new developed contribution of fluorescence nano-crystal (q-dots) as a fluorescence label for detecting pathogenic bacteria by flow cytometry (FCM) and the use of nano-magnetic particles to improve bacterial sorting by Flow cytometry cell sorting (FACS).FCM or FACS systems are based upon single cell detection by light scatter and Immunofluorescence labeling signals. The common FACS systems are based upon single or dual excitation as excitation source both for light scatter parameters and for several fluorescence detectors. Hence, for multi-labeling detection, there is a need for fluorophores with broad excitation wave length and sharp emission bands. Moreover, such fluorophores should be with high fluorescence efficiency, stable, and available for bio-molecules conjugation. Q-dots benefit from practical features which meet those -criteria. We will describe the use of q-dots as fluorescence labels for specific conjugates against Bacillus anthracis spores and Yersinia pestis bacteria, which enable the specific detection of the different species. A specific and sensitive multiplex analysis procedure for both pathogens was achieved, with high sensitivity down to 10(3) bacteria per ml in the sample.Sorting bacteria by FACS has a tremendous advantage for sensitive and selective analysis and sorting of sub-populations. However it has always been a difficult task due to the fact that bacteria are small particles (usually 1-3 µm). For such small particles, light scatter signal is on the threshold level, and many positive events may be lost. Here we will present the development of a procedure for sorting of the gram negative bacteria Y. pestis from environment samples. We will show that the application of nano-magnetic particles, as a tool for the immunomagnetic labeling and separation of the bacteria, enables fast sorting in high and low bacterial concentration down to 10 (5) cfu/ml. The nano-metric physical size of the immunospecific labeling particles disguises them from the FACS detectors; hence the bacterial population becomes the major population as opposed to being "rare events population" when using standard micro-magnetic beads for pre-enrichment.The procedure of separation and collection of bacteria enables sensitive detection and characterization methods of bacteria from complex samples.