Proteomic characterisation of leech microglia extracellular vesicles (EVs): comparison between differential ultracentrifugation and Optiprep™ density gradient isolation.

In Mammals, microglial cells are considered as the resident immune cells in central nervous system (CNS). Many studies demonstrated that, after injury, these cells are activated and recruited at the lesion site. Leech microglia present a similar pattern of microglial activation and migration upon experimental lesion of CNS. This activation is associated with the release of a large amount of extracellular vesicles (EVs). We collected EVs released by microglia primary culture and compared two different protocols of isolation: one with differential ultracentrifugation (UC) and one using an additional Optiprep™ Density Gradient (ODG) ultracentrifugation. Nanoparticles tracking analysis (NTA) and transmission electron microscopy (TEM) were used to assess vesicles size and morphology. The protein content of isolated EVs was assessed by mass spectrometry approaches. Results showed the presence of EV-specific proteins in both procedures. The extensive proteomic analysis of each single ODG fractions confirmed the efficiency of this protocol in limiting the presence of co-isolated proteins aggregates and other membranous particles during vesicles isolation. The present study permitted for the first time the characterisation of microglial EV protein content in an annelid model. Interestingly, an important amount of proteins found in leech vesicles was previously described in EV-specific databases. Finally, purified EVs were assessed for neurotrophic activity and promote neurites outgrowth on primary cultured neurons.

Real time and in vivo pharmaceutical and environmental studies with SpiderMass instrument.

Remote Infrared Matrix Assisted Laser Desorption Ionization (Remote IR MALDI) system (SpiderMass) with endogenous water as matrix allows to perform real-time DMPK in vivo. In this work, SpiderMass was used to analyze the impact on metabolite production or release of invalidated pro-protein PC1/3 macrophages by Short RNA (shRNA) versus scramble shRNA with Paclitaxel. Time course in vivo experiments were then performed on the inner and outer faces of patients' forearms or comedo treated with Melascreen (Ducray) containing ascorbyl glucoside. Finally, the impact of car pollution (emitted soot) on skin was also investigated. Taken together, we demonstrate that the SpiderMass instrument opens the door to clinical, pharmaceutical and environmental domains for real-time, in vivo pharmacokinetic (Drug Metabolism and PharmacoKinetics, DMPK) analysis.

[Immunological aspects of ovarian cancer: Therapeutic perspectives]. / Aspects immunologiques du cancer de l'ovaire : perspectives thérapeutiques.

Ovarian cancer is recognized by the immunological system of its host. Initially, it is effective to destroy and eliminate the cancer. But gradually, resistant tumor cells more aggressive and those able to protect themselves by inducing immune tolerance will be selected. Immunotherapy to be effective should consider both components of immune response with an action on cytotoxic immune effectors and action on tolerance mechanisms. The manipulations of the immune system should be cautious, because the immune effects are not isolated. A theoretically efficient handling may simultaneously cause an adverse effect which was not envisaged and could neutralize the benefits of treatment. Knowledge of tolerance mechanisms set up by the tumor is for the clinician a prerequisite before they prescribe these treatments. For each cancer, the knowledge of its immunological status is a prerequisite to propose adapted immunological therapies.

The proprotein convertase PC1/3 regulates TLR9 trafficking and the associated signaling pathways.

Endosomal TLR9 is considered as a potent anti-tumoral therapeutic target. Therefore, it is crucial to decipher the mechanisms controlling its trafficking since it determines TLR9 activation and signalling. At present, the scarcity of molecular information regarding the control of this trafficking and signalling is noticeable. We have recently demonstrated that in macrophages, proprotein convertase 1/3 (PC1/3) is a key regulator of TLR4 Myd88-dependent signalling. In the present study, we established that PC1/3 also regulates the endosomal TLR9. Under CpG-ODN challenge, we found that PC1/3 traffics rapidly to co-localize with TLR9 in CpG-ODN-containing endosomes with acidic pH. In PC1/3 knockdown macrophages, compartmentalization of TLR9 was altered and TLR9 clustered in multivesicular bodies (MVB) as demonstrated by co-localization with Rab7. This demonstrates that PC1/3 controls TLR9 trafficking. This clustering of TLR9 in MVB dampened the anti-inflammatory STAT3 signalling pathway while it promoted the pro-inflammatory NF-kB pathway. As a result, macrophages from PC1/3 KO mice and rat PC1/3-KD NR8383 macrophages secreted more pro-inflammatory cytokines such as TNF-α, IL6, IL1α and CXCL2. This is indicative of a M1 pro-inflammatory phenotype. Therefore, PC1/3 KD macrophages represent a relevant mean for cell therapy as "Trojan" macrophages.

Delivery of Alginate Scaffold Releasing Two Trophic Factors for Spinal Cord Injury Repair.

Spinal cord injury (SCI) has been implicated in neural cell loss and consequently functional motor and sensory impairment. In this study, we propose an alginate-based neurobridge enriched with/without trophic growth factors (GFs) that can be utilized as a therapeutic approach for spinal cord repair. The bioavailability of key GFs, such as Epidermal Growth factor (EGF) and basic Fibroblast Growth Factor (bFGF) released from injected alginate biomaterial to the central lesion site significantly enhanced the sparing of spinal cord tissue and increased the number of surviving neurons (choline acetyltransferase positive motoneurons) and sensory fibres. In addition, we document enhanced outgrowth of corticospinal tract axons and presence of blood vessels at the central lesion. Tissue proteomics was performed at 3, 7 and 10 days after SCI in rats indicated the presence of anti-inflammatory factors in segments above the central lesion site, whereas in segments below, neurite outgrowth factors, inflammatory cytokines and chondroitin sulfate proteoglycan of the lectican protein family were overexpressed. Collectively, based on our data, we confirm that functional recovery was significantly improved in SCI groups receiving alginate scaffold with affinity-bound growth factors (ALG+GFs), compared to SCI animals without biomaterial treatment.

Hm-MyD88 and Hm-SARM: two key regulators of the neuroimmune system and neural repair in the medicinal leech.

Unlike mammals, the CNS of the medicinal leech can regenerate damaged neurites, thus restoring neural functions after lesion. We previously demonstrated that the injured leech nerve cord is able to mount an immune response promoting the regenerative processes. Indeed neurons and microglia express sensing receptors like Hm-TLR1, a leech TLR ortholog, associated with chemokine release in response to a septic challenge or lesion. To gain insights into the TLR signaling pathways involved during these neuroimmune responses, members of the MyD88 family were investigated. In the present study, we report the characterization of Hm-MyD88 and Hm-SARM. The expression of their encoding gene was strongly regulated in leech CNS not only upon immune challenge but also during CNS repair, suggesting their involvement in both processes. This work also showed for the first time that differentiated neurons of the CNS could respond to LPS through a MyD88-dependent signalling pathway, while in mammals, studies describing the direct effect of LPS on neurons and the outcomes of such treatment are scarce and controversial. In the present study, we established that this PAMP induced the relocalization of Hm-MyD88 in isolated neurons.

Parafilm-assisted microdissection: a sampling method for mass spectrometry-based identification of differentially expressed prostate cancer protein biomarkers.

Mass spectrometry-based methods for prostate cancer biomarker discovery are hampered by their low-throughput capabilities because of extensive sample preparation. We present the parafilm-assisted microdissection technique coupled with label-free quantification and bioinformatics analysis as a means to evaluate directly protein expression changes on benign and tumor regions.

MALDI-MS and NanoSIMS imaging techniques to study cnidarian-dinoflagellate symbioses.

Cnidarian-dinoflagellate photosynthetic symbioses are fundamental to biologically diverse and productive coral reef ecosystems. The hallmark of this symbiotic relationship is the ability of dinoflagellate symbionts to supply their cnidarian host with a wide range of nutrients. Many aspects of this association nevertheless remain poorly characterized, including the exact identity of the transferred metabolic compounds, the mechanisms that control their exchange across the host-symbiont interface, and the precise subcellular fate of the translocated materials in cnidarian tissues. This lack of knowledge is mainly attributed to difficulties in investigating such metabolic interactions both in situ, i.e. on intact symbiotic associations, and at high spatial resolution. To address these issues, we illustrate the application of two in situ and high spatial resolution molecular and ion imaging techniques-matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI) and the nano-scale secondary-ion mass spectrometry (NanoSIMS) ion microprobe. These imaging techniques provide important new opportunities for the detailed investigation of many aspects of cnidarian-dinoflagellate associations, including the dynamics of cellular interactions.

[Trophoblast: conductor of the maternal immune tolerance]. / Le trophoblaste: chef d'orchestre de la tolérance immunologique maternelle.

Pregnancy is a temporary semi-allograft that survives for nine months. The importance of this event for the survival of the species justifies several tolerance mechanisms that are put into place at the beginning of pregnancy, some of which occur even at the time of implantation. The description of these mechanisms underlines the leadership of the trophoblast. The trophoblast is the conductor of the events, protects himself by expressing specific antigens and regulates the environment of the decidua according to the calendar of the events of the pregnancy The trophoblast and the decidual environment attract the effectors of immunity, almost all present in the decidua. The immunological atmosphere of the decidua evolves during the pregnancy modulating the level of activation of the immunological cells and adapting the level of activation to the stage of the pregnancy.

[Immunological analogies between ovarian cancer and pregnancy]. / Analogies immunologiques du cancer de l'ovaire et de la grossesse.

During pregnancy an environment allowing installation of tolerance toward the fetus is set up locally at the materno-fetal interface. Numerous effectors of immunity are involved in this tolerance (NK cell, T cell, Macrophages, dendritic cell). Specific mechanisms during pregnancy attract locally these immunological cells. In the decidua, they are educated toward tolerance. These mechanisms evolve during the pregnancy because at the end of the pregnancy, tolerance is broken to prepare and activate the labor. Ovarian tumors, after having surmounted the immunosurveillance, like trophoblast, chair the installation of a tolerance of their host facilitating the development of the disease. The blocking of these mechanisms of tolerance coupled with activation of mechanisms of defenses offer new perspectives in the treatment of the ovarian cancer. The authors suggest showing the analogies of the tolerance observed during ovarian cancer and pregnancy. The knowledge of the orchestration of the physiological mechanisms observed during pregnancy will offer new therapeutic targets.

[Immunological aspect of pregnancy]. / Aspects immunologiques de la grossesse.

Pregnancy is a temporary semi-allograft that survives for nine months. The importance of this event for the survival of the species justifies several tolerance mechanisms that are put into place at the beginning of pregnancy, some of which occur even at the time of implantation. The presence of multiple tolerance mechanisms and the richness of the means employed underline the central importance of the trophoblast. Understanding these mechanisms, and in particular, their integration into an overall scheme, enables the anomalies encountered in certain pathologies of pregnancy to be placed into context. Understanding these mechanisms and their interruption at the end of pregnancy should improve our understanding of disappointing results from current immunological treatments facilitate the implementation of new prophylactic and therapeutic strategies.

Polymerase chain reaction and immunoassay--matrix assisted laser desorption mass spectrometry using tag-mass technology: new tools to break down quantification limits and multiplexes.

We present a new development of the Tag-Mass concept based on a photocleavable linker with tagged molecules for polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) quantification coupled to mass spectrometry. PCR-MS and immunosorbent assay-MS with tagged oligonucleotides, bases, and antibodies will allow the acquisition of multiplexed information from genomic, transcriptomic, and proteomic experiments. This is a novel application of Tag-Mass from tissue imaging to fluid quantification and will open doors to several clinical applications ranging from biomarker-driven gene modulation to use at the patient's bedside following treatment.

On-tissue N-terminal peptide derivatizations for enhancing protein identification in MALDI mass spectrometric imaging strategies.

Matrix-assisted laser desorption/ionization (MALDI) is a new tool that can acquire the localization of various compounds, including peptides and proteins, directly from tissue sections. Despite the important developments recently performed in the field of MALDI imaging in tissue, the precise identification of compounds still needs improvement. We have developed N-terminal chemical derivatization strategies to improve tissue identification of proteins, including de novo sequencing performance. We have first focused on sulfonation agents, such as 4-SPITC and 3-SBASE. These two derivatizations were optimized to be performed directly on tissue sections. By adding a negative charge at the N-terminus of a tryptic digest peptide, we were able to generate a complete y fragment series directly from the tissue. Of these derivatizations, 3-SBASE has shown to be more efficient, as loss of the derivative group is one of the major fragmentation pathways for 4-SPITC. 3-SBASE was optimized so that the derivatization reaction could be automatically performed using an automatic microspotting device. It was then included in an automatic process that included automated trypsin digestion and matrix deposition. Derivatizations allowed the acquisition to be easily interpretable by MS(2) spectra, leading to very precise identification as well as easy manual reading of sequences for de novo sequencing. It was observed that only arginine-terminated peptides were observed after derivatization, likely due to the high gas-phase basicity of such peptides compared to those that are lysine-terminated. We also observed a stop in the y fragmentation series for peptides presenting a miscleavage. We have now begun to study a different derivatization using N-succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP). This derivatization allows the orientating of a fragmentation toward a series of fragment ions, and thus it is independent of the presence of basic residues in the sequence. This derivatization can be performed at room temperature, which greatly facilitates the automation of the process. The TMPP derivatization therefore yields an advantageous new generation of derivatives suited for use in tissue.

MALDI imaging of formalin-fixed paraffin-embedded tissues: application to model animals of Parkinson disease for biomarker hunting.

A common technique for the long-term storage of tissues in hospitals and clinical laboratories is preservation in formalin-fixed paraffin-embedded (FFPE) blocks. Such tissues stored for more than five years have not been useful for proteomic studies focused on biomarker discovery. Recently, MS-based proteomic analyses of FFPE showed positive results on blocks stored for less than 2 days. However, most samples are stored for more than one year, and thus our objective was to establish a novel strategy using as a model system 6-hydroxydopamine (6-OHDA) treated rat brain tissues stored in FFPE blocks for more than 9 years. We examined MALDI tissue profiling combining the use of automatic spotting of the MALDI matrix with in situ tissue enzymatic digestion. On adjacent sections, the identification of compounds is carried out by tissue digestion followed by nanoLC/MS-MS analysis. The combination of these approaches provides MALDI direct analysis, MALDI/MS imaging, as well as the localization of a large number of proteins. This method is validated since the analyses confirmed that ubiquitin, trans-elongation factor 1, hexokinase, and the Neurofilament M are down-regulated as previously shown in human or Parkinson animal models. In contrast, peroxidoredoxin 6, F1 ATPase, and alpha-enolase are up-regulated. In addition, we uncovered three novel putative biomarkers, the trans-elongation factor 1 (eEF1) and the collapsin response mediator 1 and 2 from protein libraries. Finally, we validate the CRMP-2 protein using immunocytochemistry and MALDI imaging based on the different ions from trypsic digestion of the protein. The access to archived FFPE tissue using MALDI profiling and imaging opens a whole new area in clinical studies and biomarker discovery from hospital biopsy libraries.

Tag-mass: specific molecular imaging of transcriptome and proteome by mass spectrometry based on photocleavable tag.

MALDI tissue imaging of tissues has become a promising technique for tracking biomarkers while determining their location and structural characterization. We have now developed specific targeting probes (oligonucleotides, antibodies), named Tag-Mass. This approach is based on probes modified with a photocleavable linker coupled with a tag cleaved and detected using mass spectrometry. Tag-Mass development is the key for a rapid, sensitive, and accurate approach to correlate levels of expression of different mRNA or proteins in diseases.

Direct analysis and MALDI imaging of formalin-fixed, paraffin-embedded tissue sections.

Formalin fixation, generally followed by paraffin embedding, is the standard and well-established processing method employed by pathologist. This treatment conserves and stabilizes biopsy samples for years. Analysis of FFPE tissues from biopsy libraries has been, so far, a challenge for proteomics biomarker studies. Herein, we present two methods for the direct analysis of formalin-fixed, paraffin-embedded (FFPE) tissues by MALDI-MS. The first is based on the use of a reactive matrix, 2,4-dinitrophenylhydrazine, useful for FFPE tissues stored less than 1 year. The second approach is applicable for all FFPE tissues regardless of conservation time. The strategy is based on in situ enzymatic digestion of the tissue section after paraffin removal. In situ digestion can be performed on a specific area of the tissue as well as on a very small area (microdigestion). Combining automated microdigestion of a predefined tissue array with either in situ extraction prior to classical nanoLC/MS-MS analysis or automated microspotting of MALDI matrix according to the same array allows the identification of both proteins by nanoLC-nanoESI and MALDI imaging. When adjacent tissue sections are used, it is, thus, possible to correlate protein identification and molecular imaging. These combined approaches, along with FFPE tissue analysis provide access to massive amounts of archived samples in the clinical pathology setting.

MALDI-MS direct tissue analysis of proteins: Improving signal sensitivity using organic treatments.

Direct tissue analysis using MALDI-MS allows the generation of profiles while maintaining the integrity of the tissue, displaying cellular localizations and avoiding tedious extraction and purification steps. However, lower spectral quality can result from direct tissue analysis due to variations in section thickness, the nature of the tissue, and the limited access to peptides/proteins due to high lipid content. To improve signal sensitivity, we have developed a tissue-washing procedure using organic solvents traditionally used for lipid extraction, i.e., CHCl3, hexane, toluene, acetone, and xylene. The increased detection for peptides/proteins (m/z 5000-30,000) is close to 40% with chloroform or xylene, and 25% with hexane, while also improving sample reproducibility for each solvent used in the present study. This strategy improved matrix cocrystallization with tissue peptides/proteins and more importantly with cytoplasmic proteins without delocalization. The extracted lipids were characterized by nanoESI-QqTOF/MS/MS using the precursor ion mode, lithium adducts, or both and were identified as phospholipids including phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and lysophosphatidylinositol, confirming membrane lipid extraction from the tissues.

Solid ionic matrixes for direct tissue analysis and MALDI imaging.

Direct analysis of tissue by MALDI-MS allows the acquisition of its biomolecular profile while maintaining the integrity of the tissue, giving cellular localization, and avoiding tedious extraction and purification steps. However, direct tissue analysis generally leads to some extent to a lowered spectral quality due to variation in thickness, freezing tissue date, and nature of the tissue. We present here new technical developments for the direct tissue analysis of peptides with ionic liquid made of matrix mixtures (alpha-cyano-4-hydroxycinnamic acid (CHCA)/2-amino-4-methyl-5-nitropyridine and alpha-cyano-4-hydroxycinnamic acid/N,N-dimethylaniline (CHCA/DANI)). The properties of these direct tissue analysis matrixes, especially CHCA/aniline when compared to CHCA, 2,5-dihydroxybenzoic acid, and sinapinic acid, are as follows: (1) better spectral quality in terms of resolution, sensitivity, intensity, noise, number of compounds detected, and contaminant tolerance, (2) better crystallization on tissues, i.e., coverage capacity, homogeneity of crystallization, homogeneity of crystal sizes, and time of crystallization, (3) better analysis duration in term of vacuum stability, (4) better resistance to laser irradiation especially for high-frequency lasers, (5) better ionic yield in negative mode, and (6) enough fragmentation yield to use the PSD mode on sections to get structural information. Applied to MALDI imaging on a MALDI LIFT-TOF with a 50-Hz laser frequency, these ionic matrixes have allowed the realization of a new type of image in both polarities and reflector mode using the same tissue section. These results give a new outlook on peptide tissue profiling by MS, characterization of compounds from tissue slices, and MALDI-MS high-quality imaging.

Characterisation of proteins differentially present in the plasma of Biomphalaria glabrata susceptible or resistant to Echinostoma caproni.

Snail immune responses towards a trematode infection are known to rely on both plasmatic and cellular host factors. As an approach to further investigate the suspected involvement of plasmatic factors in Biomphalaria glabrata resistance/susceptibility to Echinostoma caproni, we compared protein patterns of plasma collected from susceptible and resistant snails. This proteomic approach revealed that 13 plasmatic proteins exhibited significant differences in their apparent representativity. The genes corresponding to five of them were characterised by a combination of mass spectrometry and molecular cloning. They encode two isoforms of a glycolytic enzyme, two isoforms of a calcium binding protein and an inhibitor of cysteine protease. Furthermore, we investigated gene expression in parasite-exposed or -unexposed snails as well as in various tissues by quantitative PCR. This study showed that: (i) differential representation of plasma proteins between the snail strains was correlated with a differential level of transcripts; (ii) expression of these genes after parasite exposure was differentially regulated in the two strains; and (iii) these genes were expressed predominantly in the albumen gland.

Cloning, expression and pharmacological characterization of a vasopressin-related receptor in an annelid, the leech Theromyzon tessulatum.

In annelids, it has been established that arginine-vasopressin (AVP)/oxytocin (OT) superfamily peptides are involved in the maintenance of water and electrolyte homeostasis as well as reproduction. At present, there is little information on their receptors. In this study, we report the characterization of a 1.7 kb cDNA for an AVP-related receptor from the leech Theromyzon tessulatum. The open reading frame encodes a 435-aminoacid transmembrane protein that displays seven segments of hydrophobic amino acids, typical of G-protein-coupled receptors. The overall predicted protein exhibits about 30% amino-acid identities to other invertebrate, as well as vertebrate, AVP/OT receptor family members, and displays conserved characteristic features belonging to the AVP/OT receptor superfamily. RT-PCR expression experiments showed that mRNA is expressed in the genital tract, the ovary and the brain. The receptor expression is stage specific, showing a weak expression after the two first blood meals, increasing dramatically after the last blood meal during the period of sexual maturation and disappearing after egg laying. Thus, the leech AVP-related receptor may mediate reproductive functions. When expressed in COS-7 cells, the receptor binds ligands with the following rank order of potency: AVP= Arg-vasotocin >Arg-conopressin >mesotocin = OT = Lys-conopressin=isotocin>annetocin. This shows an AVP-like pharmacological profile. The transfected receptor mediates AVP-induced accumulation of inositol phosphates, indicating that the leech AVP-related receptor is functional. This study describes the characterization of a novel AVP/OT superfamily receptor in annelids, which are considered the most distant group of coelomate metazoans possessing a functional AVP/OT-related endocrine system.