Eran Zahavy

The involvement of IL-17A in the murine response to sub-lethal inhalational infection with Francisella tularensis.

Background Francisella tularensis is an intercellular bacterium often causing fatal disease when inhaled. Previous reports have underlined the role of cell-mediated immunity and IFNγ in the host response to Francisella tularensis infection. Methodology/Principal Findings Here we provide evidence for the involvement of IL-17A in host defense to inhalational tularemia, using a mouse model of intranasal infection with the Live Vaccine Strain (LVS). We demonstrate the kinetics of IL-17A production in lavage fluids of infected lungs and identify the IL-17A-producing lymphocytes as pulmonary γδ and Th17 cells. The peak of IL-17A production appears early during sub-lethal infection, it precedes the peak of immune activation and the nadir of the disease, and then subsides subsequently. Exogenous airway administration of IL-17A or of IL-23 had a limited yet consistent effect of delaying the onset of death from a lethal dose of LVS, implying that IL-17A may be involved in restraining the infection. The protective role for IL-17A was directly demonstrated by in vivo neutralization of IL-17A. Administration of anti IL-17A antibodies concomitantly to a sub-lethal airway infection with 0.1×LD50 resulted in a fatal disease. Conclusion In summary, these data characterize the involvement and underline the protective key role of the IL-17A axis in the lungs from inhalational tularemia.

Double labeling and simultaneous detection of B- and T cells using fluorescent nano-crystal (q-dots) in paraffin-embedded tissues.

A double immunohistochemical technique for the simultaneous detection of T- and B cells in paraffin-embedded mice tissues have been developed. This procedure is based on using fluorescent nano-crystals (q-dots). The benefit of using q-dots evolves from their unique fluorescence characteristics advantages: such as broad excitation spectrum, narrow emission band and high photo-bleaching threshold compare to organic fluorophores. T cells antigens (CD3) were stained using antibody-coated q-dots with max emission at 655 nm (GαRb-QD655). B cells antigens (CD45R/B220) were stained using streptavidin-coated q-dots with max emission at 585 nm (SA-QD585). The simultaneous detection of T- and B cells was demonstrated in paraffin-embedded lymph node using standard fluorescence microscope.

Application of Fluorescent Nanocrystals (q-dots) for the Detection of Pathogenic Bacteria by Flow-Cytometry

Fluorescent semiconductor nanocrystals (q-dots) benefit from practical features such as high fluorescence intensity, broad excitation band and emission diameter dependency. These unique spectroscopic characterizations make q-dots excellent candidates for new fluorescent labels in multi-chromatic analysis, such as Flow-Cytometry (FCM). In this work we shall present new possibilities of multi-labeling and multiplex analysis of pathogenic bacteria, by Flow-Cytometry (FCM) analysis and new specific IgG—q-dots conjugates. We have prepared specific conjugates against B. anthracis spores (q-dots585-IgGαB. anthracis and q-dots655-IgGαB.anthracis). These conjugates enabled us to achieve double staining of B. anthracis spores which improve the FCM analysis specificity versus control Bacillus spores. Moreover, multiplexed analysis of B. anthracis spores and Y. pestis bacteria was achieved by using specific antibodies labeled with different q-dots to obtain: q-dots585-IgGαB. anthracis and q-dots655-IgGαY.pestis, each characterized by its own emission peak as a marker. Specific and sensitive multiplex analysis for both pathogens has been achieved, down to 103 bacteria per ml in the sample.

Localized In Vivo Activation of a Photoactivatable Doxorubicin Prodrug in Deep Tumor Tissue

Sparing sensitive healthy tissue from chemotherapy exposure is a critical challenge in the treatment of cancer. The work described here demonstrates the localized in vivo photoactivation of a new chemotherapy prodrug of doxorubicin (DOX). The DOX prodrug (DOX‐PCB) was 200 times less toxic than DOX and was designed to release pure DOX when exposed to 365 nm light. This wavelength was chosen because it had good tissue penetration through a 1 cm diameter tumor, but had very low skin penetration, due to melanin absorption, preventing uncontrolled activation from outside sources. The light was delivered specifically to the tumor tissue using a specialized fiber‐optic LED system. Pharmacokinetic studies showed that DOX‐PCB had an α circulation half‐life of 10 min which was comparable to that of DOX at 20 min. DOX‐PCB demonstrated resistance to metabolic cleavage ensuring that exposure to 365 nm light was the main mode of in vivo activation. Tissue extractions from tumors exposed to 365 nm light in vivo showed the presence of DOX‐PCB as well as activated DOX. The exposed tumors had six times more DOX concentration than nearby unexposed control tumors. This in vivo proof of concept demonstrates the first preferential activation of a photocleavable prodrug in deep tumor tissue.

Nano-biotechnology for biomedical and diagnostic research

Preface.- 1. Biomolecule/Nanomaterial Hybrid Systems for Nanobiotechnology.- 1.1. Electrical Contacting of Enzymes with Electrodes for the Construction of Amperometric Biosensors and Biofuel Cells.- 1.2. Catalytic Properties of Metalic Nanoparticles (NPs) and their Implementation for Sensing and Nanocircuitry.- 1.3. Bioanalytical Applications of Hybrid Semiconductor-Protein Systems.- 1.4. Biomolecules as Templates for Nanoscale Circuitry.- 1.5. Conclusions and Perspectives.- 2. Superresolution Optical Flunctuation Imaging.- 2.1. Main article.- 2.2. Conclusion.- 3. Application of Nanoparticles for the Detection and Sorting of Pathogenic Bacteria by Flow-Cytometry.- 3.1. Introduction.- 3.2. Results.- 3.3. Conclusion.- 4. Advancing Nanostructured Porous Si-Based Optical Transducers for Label Free Bacteria Detection.- 4.1. Introduction.- 4.2. Materials and Methods.- 4.3. Results and Discussion.- 4.4. Conclusions.- 5. Gold Fibers as a Platform for Biosensing.- 5.1. Introduction.- 5.2. Experimental.- 5.3. Results and Discussion.- 5.4. Electrochemical Evaluation of Gox on Gold Fibers.- 5.5. Summary and Conclusions.- 6. Surface-Enhanced Raman Spectroscopy of Organic Molecules Adsorbed on Metallic Nanoparticles.- 6.1. Introduction.- 6.2. Experimental.- 6.3. Results and Discussion.- 6.4. Summary and Conclusions.- 7. Quantum Dots and Fluorescent Protein FRET-based Biosensors.- 7.1. Introduction.- 7.2. Materials and Methods.- 7.3. Results.- 7.4. Discussion.- 8. Semiconductor Quantum Dots as FRET-Acceptors for Multiplexed Diagnostics and Molecular Ruler Application.- 8.1. Introduction.- 8.2. Short Theoretical and Practical Background.- 8.3. Applications.- 8.4. Conclusions and Outlook.- 9. Assembly and Microscopic Characterization of DNA Origami Structures.- 9.1. Introduction.- 9.2. DNA as a Material for ?Molecular Self-Assembly.- 9.3. Modification of Origami Structures.- 9.4. Characterization of DNA Origami Structures.- 9.5. Conclusion and Outlook.- 10. DNA Nanotechnology.- 10.1. DNA Nanostructures for Amplified Sensing.- 10.2. Ultrasensitive Detection of DNA through Isothermal Replication Processes using DNA Enzymes.- 10.3. Programmed Nanostructures Acting as DNA Machines.- 10.4. Self-Assembly of Functional DNA-Protein Nanostructures.- 10.5. Conclusions and Perspectives.- 11. Role of Carbohydrate (lectin) receptors in the Macrophage Uptake of Dextran-Coated Iron Oxide Nanoparticles.- 11.1. Introduction.- 11.2. Materials and Methods.- 11.3. Results and Discussion.- 11.4. Conclusions.- 12. Toxicity of Gold Nanoparticles on Somatic and Reproductive Cells.- 12.1. Introduction.- 12.2. Effect of Gold Nanoparticles on Somatic Cells.- 12.3. Reproductive Toxicology of Gold Nanoparticles.- 12.4. Conclusion.-13. Ultrasound Activated Nano-Encapsulated Targeted Drug Delivery and Tumour Cell Poration.- 13.1. Introduction.- 13.2. Materials and Methods.- 13.3. Results.- 13.4. Discussion.- 13.5. Future Work.- 14. Ultrasound Mediated Localized Drug Delivery.- 14.1. Introduction.- 14.2. Ultrasound Intensity Level of 1.5 MPa.- 14.3. Ultrasound Intensity Levels Below 1 MPa.- 14.4. Localized SHERPA Activation.- 14.5. Discussion.- 14.6. Conclusions.- 15. Sonochemical Proteinaceous Microspheres for Wound Healing.- 15.1. Introduction.- 15.2. Materials and Methods.- 15.3. Results and Discussion.- 15.4. Conclusions.- 16. Alendronate Liposomes for Antitumor Therapy: Activation of gammadelta T Cells and Inhibition of Tumor Growth.- 16.1. Introduction.- 16.2. Materials and Methods.- 16.3. Results.- 16.4. Discussion.

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.

The search for early markers of plague: evidence for accumulation of soluble Yersinia pestis LcrV in bubonic and pneumonic mouse models of disease.

Markers of the early stages of plague, a rapidly progressing deadly disease, are crucial for enabling the onset of an effective treatment. Here, we show that V-antigen protein (LcrV) is accumulated in the serum of Yersinia pestis-infected mice before bacterial colonization of the spleen and dissemination to blood, in a model of bubonic plague. LcrV accumulation is detected earlier than that of F1 capsular antigen, an established marker of disease. In a mouse model of pneumonic plague, LcrV can be determined in the bronchoalveolar lavage fluid somewhat later than F1, but before dissemination of Y. pestis to the blood. Thus, determination of soluble LcrV is suggested as a potential useful tool for monitoring disease progression in both bubonic and pneumonic plague. Moreover, it may be of particular advantage in cases of infections with F1 nonproducing strains.

Enrichment of Yersinia pestis from Blood Cultures Enables Rapid Antimicrobial Susceptibility Determination by Flow Cytometry

Mortality from plague is high if not treated with the proper antibiotics within 18-24 hours after onset of symptoms. The process of antibiotic susceptibility determination of Yersinia pestis isolated from blood samples may extend from 4 to more than 7 days, since the in vitro growth is very slow. To accelerate this process, we developed an enrichment protocol as well as a non-standard yet reliable method for rapid antibiotic susceptibility analysis of Y. pestis from blood cultures using flow cytometry technology. This rapid method is applicable to blood cultures containing low levels of Y. pestis.

Extraction Protocol and Mass Spectrometry Method for Quantification of Doxorubicin Released Locally from Prodrugs in Tumor Tissue

The localized conversion of inactive doxorubicin prodrug chemotherapeutics to pharmacalogically active forms is difficult to quantify in mouse tumor models because it occurs only in small regions of tissue. The tumor tissue extraction protocol and LC-MS/MS analysis method described here were optimized to obtain a detection limit of 7.8 pg for the activated doxorubicin and 0.36 ng for the doxorubicin prodrug. This method can be useful for determining the biodistribution and activation efficiency for many different doxorubicin prodrugs. It can also be used for quantification of doxorubicin from tumor models that have poor vascularization resulting in low tissue accumulation.

Bacterial viability assessment by flow cytometry analysis in soil

Soil microhabitats and their heterogeneity are often considered to be among the most important factors affecting soil biotic communities. The microbial community has become one of the most important links in soil nutrient cycles and trophic components due to its role in biological processes, spatial and temporal dynamics, and physiological adaptation. Sandy-soil desert systems are characterized by fast water infiltration during the rainy season, high salinity, and low moisture availability in the upper soil layers. Plants have developed different ecophysiological adaptations in order to cope with this harsh environment. The Tamarix aphylla is known to be one of the most commonly adapted plants, exhibiting a mechanism for secretion of excess salts as aggregates through its leaves. These leaves aggregate beneath the plant, creating ‘islands of salinity’. Soil biotic components are, therefore, exposed to extreme abiotic stress conditions in this niche. The goal of this study was to examine the effect of T. aphylla on the live/dead bacterial population ratio on a spatial and temporal scale. The results emphasize the effect of abiotic factors, which changed on temporal as well as spatial scales, and also on the size of the active soil bacterial community, which fluctuated between 1.44% and 25.4% in summer and winter, respectively. The results of this study elucidate the importance of moisture availability and the ‘island-of-salinity’ effect on the active microbial community in a sandy desert system.