Two-photon intravital imaging of lungs during anthrax infection reveals long-lasting macrophage-dendritic cell contacts.

International audience: Dynamics of the lung immune system at a microscopic level are largely unknown because of inefficient methods to rid chest motion during image acquisition. In this study, we developed an improved intravital method for two-photon lung imaging uniquely based on a posteriori parenchymal tissue motion correction. We took advantage of the alveolar collagen pattern given by second harmonic generation signal as a reference for frame registration. We describe here for the first time a detailed dynamic account of two major lung immune cell populations, alveolar macrophages and CD11b-positive dendritic cells, during homeostasis and infection by spores of Bacillus anthracis, the agent of anthrax. We show that after alveolar macrophages capture spores, CD11b-positive dendritic cells come in prolonged contact with infected macrophages. Dendritic cells are known to carry spores to the draining lymph nodes and elicit the immune response in pulmonary anthrax. The intimate and long-lasting contacts between these two lines of defense may therefore coordinate immune responses in the lung through an immunological synapse-like process

Fluctuation of the SP/non-SP phenotype in the C6 glioma cell line

AbstractUsing the C6 glioma cell as a paradigm, we found that (i) the clonogenicity of C6 cells is several orders of magnitude higher than the percentage of SP cells; (ii) non-SP cells are able to generate SP cells, and conversely SP cells generate non-SP cells; (iii) non-SP sorted cells behave as tumorigenic cells. Hence, in C6 cells cultured in serum-containing medium, SP cells can be generated from non-SP cells. This dynamic equilibrium explains in C6 cells the maintenance of the SP phenotype with cell passaging and demonstrates the existence of tumorigenic non-SP cells

Cytometric assessment of mitochondria using fluorescent probes.

International audienceMitochondria are most important organelles in the survival of eukaryotic aerobic cells because they are the primary producers of ATP, regulators of ion homeostasis or redox state, and producers of free radicals. The key role of mitochondria in the generation of primordial ATP for the survival and proliferation of eukaryotic cells has been proven by extensive biochemical studies. In this context, it is crucial to understand the complexity of the mitochondrial compartment and its functionality and to develop experimental tools allowing the assessment of its nature and its function and metabolism. This review covers the role of the mitochondria in the cell, focusing on its structure, the mechanism of the mitochondrial respiratory chain, the maintenance of the transmembrane potential and the production of reactive oxygen species. The main probes used for mitochondrial compartment monitoring are described. In addition, various applications using mitochondrial-specific probes are detailed to illustrate the potential of flow and image cytometry in the study of the mitochondrial compartment. This review contains a panel of tools to explore mitochondria and to help researchers design experiments, determine the approach to be employed, and interpret their results

Thematic workshop on fluorescence compensation settings in multicolor flow cytometry.

International audienceIn his program of thematic one-day workshops, the French Association of Cytometry had organized a workshop dedicated to the fluorescence compensation settings in multicolor flow cytometry. This special day was in honor of our past President Jean Luc D'Hautcourt who has been involved in the quality of the use of flow cytometry in its clinical and research purposes. Review on fluorescence phenomena, compensation rules, settings, and few observed confounding situations were presented

Vagus nerve stimulation: from epilepsy to the cholinergic anti-inflammatory pathway.

International audienceBACKGROUND: The brain and the gut communicate bidirectionally through the autonomic nervous system (ANS). The vagus nerve (VN), a major component of the ANS, plays a key role in the neuro-endocrine-immune axis to maintain homeostasia through its afferents (through the activation of the hypothalamic pituitary adrenal axis and the central ANS) and through its efferents (i.e. the cholinergic anti-inflammatory pathway; CAP). The CAP has an anti-TNF effect both through the release of acetylcholine at the distal VN acting on macrophages and through the connection of the VN with the spleen through the splenic sympathetic nerve. Vagus nerve stimulation (VNS) of vagal afferents at high frequency (20-30 Hz) is used for the treatment of drug-resistant epilepsy and depression. Low-frequency (5 Hz) VNS of vagal efferents activates the CAP for an anti-inflammatory effect that is as an anti-TNF therapy in inflammatory diseases were TNF is a key cytokine as represented by experimental sepsis, postoperative ileus, burn-induced intestinal barrier injury, colitis. However, both vagal afferents and efferents are activated by VNS. PURPOSE: The objective of this review was to explore the following: (i) the supporting evidence for the importance of VNS in epilepsy (and depression) and its mechanisms of action, (ii) the anti-inflammatory characteristics of the VN, (iii) the experimental evidence that VNS impact on inflammatory disorders focusing on the digestive tract, and (iv) how VNS could potentially be harnessed therapeutically in human inflammatory disorders such as inflammatory bowel diseases, irritable bowel syndrome, postoperative ileus, rheumatoid arthritis as an anti-inflammatory therapy

Lack of evidence of sustained hematopoietic reconstitution after transplantation of unmanipulated adult liver stem cells in monkeys.

International audienceThe aim of this study was to search for hematopoietic potential in the liver of non-human primates. Lethally irradiated (2 x 5 Gy gamma) macaque monkeys were given autologous hepatic mononuclear cells (HMNC) isolated from a liver lobe by perfusion and digestion with 0.1% collagenase. Two monkeys were given intramedullary injections of HMNC (18.6 x 10(6)/kg, 20.4 x 10(6)/kg) and two others were co-transplanted with HMNC (14.35 x 10(6)/kg, 96.5 x 10(6)/kg) and bone marrow mesenchymal stem cells (0.42 x 10(6)/kg, 1.16 x 10(6)/kg). All monkeys exhibited a transient neutrophil recovery from day 22 for 10 days, but failed to produce platelets and remained transfusion-dependent. In conclusion, adult liver stem cells from a monkey model show a low level of in vivo hematopoietic potential, suggesting ex vivo manipulation will be required before clinical use of such cells

Flow Cytometry Approach to Quantify the Viability of Milk Somatic Cell Counts after Various Physico-Chemical Treatments.

Flow cytometry has been used as a routine method to count somatic cells in milk, and to ascertain udder health and milk quality. However, few studies investigate the viability of somatic cells and even fewer at a subpopulation level to follow up how the cells can resist to various stresses that can be encountered during technological processes. To address this issue, a flow cytometry approach was used to simultaneously identify cell types of bovine milk using cell-specific antibodies and to measure the cell viability among the identified subpopulations by using a live/dead cell viability kit. Confirmation of the cell viability was performed by using conventional microscopy. Different physico-chemical treatments were carried out on standardized cell samples, such as heat treatment, various centrifugation rates and storage in milk or in PBS pH 7.4 for three days. Cytometry gating strategy was developed by using blood cell samples stored at 4°C in PBS and milk cell samples heat-treated at 80°C for 30 min as a control for the maximum (95.9%) and minimum (0.7%) values of cell viability respectively. Cell viability in the initial samples was 39.5% for all cells and varied for each cell population from 26.7% for PMNs, to 32.6% for macrophages, and 58.3% for lymphocytes. Regarding the physico-chemical treatments applied, somatic cells did not sustain heat treatment at 60°C and 80°C in contrast to changes in centrifugation rates, for which only the higher level, i.e. 5000×g led to a cell viability decrease, down to 9.4%, but no significant changes within the cell subpopulation distribution were observed. Finally, the somatic cells were better preserved in milk after 72h storage, in particular PMNs, that maintained a viability of 34.0 ± 2.9% compared to 4.9±1.9% in PBS, while there was almost no changes for macrophages (41.7 ± 5.7% in milk vs 31.2 ± 2.4% in PBS) and lymphocytes (25.3 ± 3.0% in milk vs 11.4 ± 3.1% in PBS). This study provides a new array to better understand milk cell biology and to establish the relationship between the cell viability and the release of their endogenous enzymes in dairy matrix

Mesenchymal stem cells rescue CD34+ cells from radiation-induced apoptosis and sustain hematopoietic reconstitution after coculture and cografting in lethally irradiated baboons: is autologous stem cell therapy in nuclear accident settings hype or reality?

International audienceAutologous stem cell therapy (ACT) has been proposed to prevent irradiated victims from bone marrow (BM) aplasia by grafting hematopoietic stem and progenitor cells (HSPCs) collected early after damage, provided that a functional graft of sufficient size could be produced ex vivo. To address this issue, we set up a baboon model of cell therapy in which autologous peripheral blood HSPCs collected before lethal total body irradiation were irradiated in vitro (2.5 Gy, D0 1 Gy) to mimic the cell damage, cultured in small numbers for a week in a serum-free medium in the presence of antiapoptotic cytokines and mesenchymal stem cells (MSCs) and then cografted. Our study shows that baboons cografted with expanded cells issued from 0.75 and 1 x 10(6)/kg irradiated CD34+ cells and MSCs (n=2) exhibited a stable long-term multilineage engraftment. Hematopoietic recovery became uncertain when reducing the CD34+ cell input (0.4 x 10(6)/kg CD34+ cells; n=3). However, platelet recovery was accelerated in all surviving cografted animals, when compared with baboons transplanted with unirradiated, unmanipulated CD34+ cells (0.5-1 x 10(6)/kg, n=4). Baboons grafted with MSCs alone (n=3) did not recover. In all cases, the nonhematopoietic toxicity remained huge. This baboon study suggests that ACT feasibility is limited

Impact of Anesthetics on Immune Functions in a Rat Model of Vagus Nerve Stimulation

Vagus nerve stimulation (VNS) has been successfully performed in animals for the treatment of different experimental models of inflammation. The anti-inflammatory effect of VNS involves the release of acetylcholine by vagus nerve efferent fibers inhibiting pro-inflammatory cytokines (e.g. TNF-a) produced by macrophages. Moreover, it has recently been demonstrated that splenic lymphocytic populations may also be involved. As anesthetics can modulate the inflammatory response, the current study evaluated the effect of two different anesthetics, isoflurane and pentobarbital, on splenic cellular and molecular parameters in a VNS rat model. Spleens were collected for the characterization of lymphocytes subpopulations by flow cytometry and quantification of cytokines secretion after in vitro activation. Different results were observed depending on the anesthetic used. The use of isoflurane displayed a non-specific effect of VNS characterized by a decrease of most splenic lymphocytes sub-populations studied, and also led to a significantly lower TNF-a secretion by splenocytes. However, the use of pentobarbital brought to light immune modifications in non-stimulated animals that were not observed with isoflurane, and also revealed a specific effect of VNS, notably at the level of T lymphocytes ’ activation. These differences between the two anesthetics could be related to the anti-inflammatory properties of isoflurane. In conclusion, pentobarbital is more adapted than isoflurane in the study of the anti-inflammatory effect of VNS on a