The microenvironment of DLBCL is characterized by noncanonical macrophages recruited by tumor-derived CCL5.

Tissue invasion by tumor cells induces a host inflammatory response that variably impacts tumorigenesis. This has been well documented for tumor-associated macrophages (TAMs) that could play a pro/M2- or an anti/M1-tumoral function. TAMs frequently infiltrate diffuse large B-cell lymphoma (DLBCL), an aggressive neoplasm arising from germinal center-experienced B cells. However, the pathway leading to the presence of TAMs in DLBCL remains unknown, and their impact is unclear. Here, we show that some DLBCL tumor cells expressed the chemokine CCL5, enabling the differential recruitment of blood monocytes through their expression of CCR1 and CCR5. CCL5 expression by DLBCL was not related to molecular subtypes, and healthy tonsillar B cells did not produce this chemokine, implying a posttransformation event. A single-cell analysis revealed that most DLBCL TAMs had a noncanonical gene signature with the concomitant expression of M1 and M2 genes. The presence of noncanonical TAMs may explain the lack of impact of macrophages on DLBCL development reported in some survival studies.

Targeting self and neo-epitopes with a modular self-adjuvanting cancer vaccine.

Induction of a potent CD4 and CD8 T-cell response against tumor-specific and tumor-associated antigen is critical for eliminating tumor cells. Recent vaccination strategies have been hampered by an inefficacious and low amplitude immune response. Here we describe a self-adjuvanted chimeric protein vaccine platform to address these challenges, characterized by a multidomain construction incorporating (i) a cell penetrating peptide (CPP) allowing internalization of several multiantigenic Major Histocompatibility Complex (MHC)-restricted peptides within (ii) the multiantigenic domain (Mad) and (iii) a TLR2/4 agonist domain (TLRag). Functionality of the resulting chimeric protein is based on the combined effect of the above-mentioned three different domains for simultaneous activation of antigen presenting cells and antigen cross-presentation, leading to an efficacious multiantigenic and multiallelic cellular immune response. Helper and cytotoxic T-cell responses were observed against model-, neo- and self-antigens, and were highly potent in several murine tumor models. The safety and the immunogenicity of a human vaccine candidate designed for colorectal cancer treatment was demonstrated in a non-human primate model. This newly engineered therapeutic vaccine approach is promising for the treatment of poorly infiltrated tumors that do not respond to currently marketed immunotherapies.

CXCL-8/IL8 Produced by Diffuse Large B-cell Lymphomas Recruits Neutrophils Expressing a Proliferation-Inducing Ligand APRIL.

Tumor-infiltrating neutrophils have been implicated in malignant development and progression, but mechanisms are ill defined. Neutrophils produce a proliferation-inducing ligand APRIL/TNFSF13, a factor that promotes development of tumors from diverse origins, including diffuse large B-cell lymphoma (DLBCL). High APRIL expression in DLBCL correlates with reduced patient survival, but the pathway(s) dictating APRIL expression are not known. Here, we show that all blood neutrophils constitutively secrete APRIL, and inflammation-associated stimuli, such as TNF, further upregulate APRIL. In a significant fraction of DLBCL patients, tumor cells constitutively produced the ELC-CXC chemokine CXCL-8 (IL8), enabling them to recruit APRIL-producing blood neutrophils. CXCL-8 production in DLBCL was unrelated to the cell of origin, as APRIL-producing neutrophils infiltrated CXCL-8+ DLBCL from both germinal center (GC) and non-GC subtypes. Rather, CXCL-8 production implied events affecting DNA methylation and acetylation. Overall, our results showed that chemokine-mediated recruitment of neutrophils secreting the tumor-promoting factor APRIL mediates DLBCL progression. Cancer Res; 77(5); 1097-107. ©2016 AACR.

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.

Experimental Quantification of Delayed Radiation-Induced Organ Damage in Highly Irradiated Rats With Bone Marrow Protection: Effect of Radiation Dose and Organ Sensitivity.

The evolution of organ damage following extensive high-dose irradiation remains largely unexplored and needs further investigation. Wistar rats [with or without partial bone marrow protection (∼20%)] were irradiated at lethal gamma-ray doses (12, 14, and 16 Gy) and received antibiotic support. While total-body-irradiated rats did not survive, bone marrow protection (achieved by protecting hind limbs behind a lead wall) combined with antibiotic support allowed survival of 12-Gy and 14-Gy irradiated rats for more than 3 mo, with a late phase of body weight loss and altered clinical status. Histological analysis of radiation-induced damages in visceral organs (liver, kidney, and ileum), performed 64 and 104 d after high-dose body irradiation, indicates that the extent and the evolution of damage depend on both the irradiation dose and organ. A dose-related aggravation of lesions was observed in the liver and kidney but not in the ileum. In contrast to the liver, alterations in the kidney and ileum aggravate with time, emphasizing the need to develop new efficient countermeasures to protect both the gastrointestinal tract and kidney from late-occurring radiation effects. Specifically, the complex evolution of organ damage presented in this paper offers the possibility to explore and then validate specific therapeutic windows using candidate drugs targeted to each injured visceral organ.

Gadolinium nanoparticles and contrast agent as radiation sensitizers.

The goal of the present study was to evaluate and compare the radiosensitizing properties of gadolinium nanoparticles (NPs) with the gadolinium contrast agent (GdCA) Magnevist(®) in order to better understand the mechanisms by which they act as radiation sensitizers. This was determined following either low energy synchrotron irradiation or high energy gamma irradiation of F98 rat glioma cells exposed to ultrasmall gadolinium NPs (GdNPs, hydrodynamic diameter of 3 nm) or GdCA. Clonogenic assays were used to quantify cell survival after irradiation in the presence of Gd using monochromatic x-rays with energies in the 25 keV-80 keV range from a synchrotron and 1.25 MeV gamma photons from a cobalt-60 source. Radiosensitization was demonstrated with both agents in combination with X-irradiation. At the same concentration (2.1 mg mL(-1)), GdNPS had a greater effect than GdCA. The maximum sensitization-enhancement ratio at 4 Gy (SER4Gy) was observed at an energy of 65 keV for both the nanoparticles and the contrast agent (2.44 ± 0.33 and 1.50 ± 0.20, for GdNPs and GdCA, respectively). At a higher energy (1.25 MeV), radiosensitization only was observed with GdNPs (1.66 ± 0.17 and 1.01 ± 0.11, for GdNPs and GdCA, respectively). The radiation dose enhancements were highly 'energy dependent' for both agents. Secondary-electron-emission generated after photoelectric events appeared to be the primary mechanism by which Gd contrast agents functioned as radiosensitizers. On the other hand, other biological mechanisms, such as alterations in the cell cycle may explain the enhanced radiosensitizing properties of GdNPs.

The extent of irradiation-induced long-term visceral organ damage depends on cranial/brain exposure.

In case of high-dose radiation exposure, mechanisms controlling late visceral organ damage are still not completely understood and may involve the central nervous system. To investigate the influence of cranial/brain irradiation on late visceral organ damage in case of high-dose exposure, Wistar rats were irradiated at 12 Gy, with either the head and fore limbs or the two hind limbs protected behind a lead wall (head- and hind limbs-protected respectively), which allows long-term survival thanks to bone marrow protection. Although hind limbs- and head-protected irradiated rats exhibited similar hematopoietic and spleen reconstitution, a late body weight loss was observed in hind limbs-protected rats only. Histological analysis performed at this time revealed that late damages to liver, kidney and ileum were attenuated in rats with head exposed when compared to animals whose head was protected. Plasma measurements of inflammation biomarkers (haptoglobin and the chemokine CXCL1) suggest that the attenuated organ damage in hind limbs-protected rats may be in part related to reduced acute and chronic inflammation. Altogether our results demonstrate the influence of cranial/brain exposure in the onset of organ damage.

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

The dynamics of the lung immune system at the microscopic level are largely unknown because of inefficient methods of restraining 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 the 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.

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-α) 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 sub-populations 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-α 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 an anesthetized rat model in that it allows more accurate monitoring of subtle immunomodulatory processes.

Assessment of total- and partial-body irradiation in a baboon model: preliminary results of a kinetic study including clinical, physical, and biological parameters.

This biodosimetry study used irradiated baboons to investigate the efficacy of a kinetic multiparameter (clinical, physical, and biological) approach for discriminating partial-body irradiation (PBI) and total-body irradiation (TBI). Animals were unilaterally (front) exposed to 60Co gamma rays (8 to 32 cGy min) using either TBI or vertical left hemi-body irradiation (HBI), as follows: 2.5 Gy TBI (n = 2), 5 Gy TBI (n = 2), 5 Gy HBI (n = 2), and 10 Gy HBI (n = 2). Midline tissue doses were measured at the anterior iliac crest level with an ionization chamber, and body dosimetry was performed using thermoluminescent dosimeters. Blood samples were collected before exposure and from 1 h until 200 d after irradiation. Clinical status, complete blood cell count, biochemical parameters, and cytogenetic analysis were evaluated. The partial least square discriminant analysis chosen for statistical analysis showed that the four groups of irradiated baboons were clearly separated. However, the dicentric chromosome assay may not distinguish HBI from TBI in confounding situations where equivalent whole-body doses are similar and the time of exposure is sufficient for peripheral blood lymphocyte homogenization. Interestingly, as bone marrow shielding in HBI animals prevented aplasia from happening, hematologic parameters such as the platelet count and Flt-3 ligand level helped to distinguish HBI and TBI. Moreover, the ratio of neutrophil to lymphocyte counts, creatine kinase, and citrulline levels may be discriminating biomarkers of dose or injury. Both early and delayed clinical signs and bioindicators appear to be useful for assessment of heterogeneous irradiation.

Cytometric assessment of mitochondria using fluorescent probes.

Mitochondria 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.

Optimal epitope composition after antigen screening using a live bacterial delivery vector: application to TRP-2.

Immunotherapeutic approaches, based on the generation of tumor-specific cytotoxic T-lymphocytes (CTL), are currently emerging as promising strategies of anti-tumor therapy. The potential use of attenuated bacteria as engineered vectors for vaccine development offers several advantages, including the stimulation of innate immunity. We developed an attenuated live bacterial vector using the type III secretion system (TTSS) of Pseudomonas aeruginosa to deliver in vivo tumor antigens. Using an inducible and rapid expression plasmid, vaccination with several antigens of different length and epitope composition, including TRp-2, gp100 and MUC18, was evaluated against glioma tumor cells. We observed similar CTL immunity and T-cell receptor (TCR) repertoire diversity with the vaccines, TRP2(125-243), TRP2L(125-376) and TRP2S(291-376). However, only immunization with TRP2L(125-376) induced significant anti-tumor immunity. Taken together, our data indicate the importance of the epitopes composition and/or peptide length of these peptides for inducing cytotoxic T-lymphocyte (CTL) mediated immunity. Characteristics that consistently improved anti-tumor immunity include: long peptides with immunodominant and cryptic CD8(+) epitopes, and strong CD4(+) Th epitopes. Our bacterial vector is versatile, easy-to-use and quick to produce. This vector is suitable for rapid screening and evaluation of antigens of varying length and epitope composition.

Characterisation of normal and cancer stem cells: one experimental paradigm for two kinds of stem cells.

The characterisation of normal stem cells and cancer stem cells uses the same paradigm. These cells are isolated by a fluorescence-activated cell sorting step and their stemness is assayed following implantation into animals. However, differences exist between these two kinds of stem cells. Therefore, the translation of the experimental procedures used for normal stem cell isolation into the research field of cancer stem cells is a potential source of artefacts. In addition, normal stem cell therapy has the objective of regenerating a tissue, while cancer stem cell-centred therapy seeks the destruction of the cancer tissue. Taking these differences into account is critical for anticipating problems that might arise in cancer stem cell-centred therapy and for upgrading the cancer stem cell paradigm accordingly.

Cytokines in combination to treat radiation-induced myelosuppresssion: evaluation of SCF + glycosylated EPO + pegylated G-CSF as an emergency treatment in highly irradiated monkeys.

Multicytokine therapy may be useful to counteract radiation-induced myelosuppression. We assessed the stem cell factor + glycosylated erythropoietin + pegylated granulocyte colony-stimulating factor combination (SEG) as an emergency treatment. SEG in highly irradiated monkeys efficacy appeared to be restricted to granulopoiesis. Early administration of Erythropoietin did not prevent radiation-induced anemia.

Effects of Hoechst 33342 on C2C12 and PC12 cell differentiation.

Accumulative evidence demonstrates that normal as well as cancer stem cells can be identified as a side population following Hoechst 33342 staining and flow cytometric analysis. This popular method is based on the ability of stem cells to efflux this fluorescent vital dye. We demonstrate that Hoechst 33342 can affect cell differentiation, suggesting potential complications in the interpretation of data.

Antiapoptotic cytokines in combination with pegfilgrastim soon after irradiation mitigates myelosuppression in nonhuman primates exposed to high irradiation dose.

OBJECTIVE: Preservation of hematopoietic stem and progenitor cells from early radiation-induced apoptosis is the rationale for emergency antiapoptotic cytokine therapy (EACK) after radiation accidents. This strategy is based on the combination of stem cell factor + Flt3-ligand + thrombopoietin + interleukin 3 (SFT3). The long-term safety and efficacy of EACK in managing severe radiation exposure were evaluated. MATERIAL AND METHODS: Early administration of SFT3 + pegfilgrastim was assessed in 7-Gy gamma total body-irradiated (TBI) monkeys. Efficiency of delayed administration was also addressed after 5-Gy TBI. RESULTS: Here we showed that a single, intravenous injection of SFT3 2 hours after 7-Gy TBI reduced the period of thrombocytopenia (platelet count <20 x 10(9)/L: 0.8 +/- 1.5 day vs 23.8 +/- 15.9 days in controls; p < 0.05) and blood transfusion needs. Moreover, addition of pegfilgrastim to SFT3 treatment shortened the period of neutropenia compared with SFT3 and control groups (neutrophil count <0.5 x 10(9)/L: 7 +/- 1.4 days vs 13 +/- 3.2 days and 15.2 +/- 1.5 days; p < 0.05). In both SFT3 groups, bone marrow activity recovered earlier and, in contrast with controls, platelet count returned to baseline values from 250 days after irradiation. Furthermore, delayed (48 hours) single SFT3 administration in 5-Gy irradiated monkeys significantly reduced thrombocytopenia compared to controls. Finally, SFT3 did not increase frequency of total chromosome translocations observed in the blood lymphocytes of controls 1 year after 5 Gy TBI. CONCLUSION: These results suggest the safety and efficacy of EACK in managing severe radiation exposure.

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

Using 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.

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

The 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.

Thematic workshop on fluorescence compensation settings in multicolor flow cytometry.

In 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.

Which place for stem cell therapy in the treatment of acute radiation syndrome?

Radiation-induced (RI) tissue injuries can be caused by radiation therapy, nuclear accidents or radiological terrorism. Notwithstanding the complexity of RI pathophysiology, there are some effective approaches to treatment of both acute and chronic radiation damages. Cytokine therapy is the main strategy capable of preventing or reducing the acute radiation syndrome (ARS), and hematopoietic growth factors (GF) are particularly effective in mitigating bone marrow (BM) aplasia and stimulating hematopoietic recovery. However, first, as a consequence of RI stem and progenitor cell death, use of cytokines should be restricted to a range of intermediate radiation doses (3 to 7 Gy total body irradiation). Second, ARS is a global illness that requires treatment of damages to other tissues (epithelial, endothelial, glial, etc.), which could be achieved using pleiotropic or tissue-specific cytokines. Stem cell therapy (SCT) is a promising approach developed in the laboratory that could expand the ability to treat severe radiation injuries. Allogeneic hematopoietic stem cell transplantation (BM, mobilized peripheral blood and cord blood) transplantation has been used in radiation casualties with variable success due to limiting toxicity related to the degree of graft histocompatibility and combined injuries. Ex vivo expansion should be used to augment cord blood graft size and/or promote very immature stem cells. Autologous SCT might also be applied to radiation casualties from residual hematopoietic stem and progenitor cells (HSPC). Stem cell plasticity of different tissues such as liver or skeletal muscle, may also be used as a source of hematopoietic stem cells. Finally, other types of stem cells such as mesenchymal, endothelial stem cells or other tissue committed stem cells (TCSC), could be used for treating damages to nonhematopoietic organs.