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Emerging gene therapy clinical trials test the correction of hemophilia A (HA) by replacing factor VIII (FVIII) in autologous hematopoietic stem cells (HSCs). Although it is known that platelets, monocyte/macrophages, and mesenchymal stromal cells can secrete transgenic FVIII, a systematic examination of blood lineages as extrahepatic sources of FVIII, to our knowledge, has not yet been performed. In this study, we sought to provide a comprehensive map of native and lentivirus-based transgenic FVIII production from HSC stage to mature blood cells, through a flow cytometry analysis. In addition, we generated a model of transient HA in zebrafish based on antisense RNA, to assess the corrective potential of the FVIII-transduced HSCs. We discovered that FVIII production begins at the CD34+ progenitor stage after cytokine stimulation in culture. Among all mature white blood cells, monocytes are the largest producers of native FVIII and can maintain protein overexpression during differentiation from HSCs when transduced by a FVIII lentiviral vector. Moreover, the addition of the HSC self-renewal agonist UM171 to CD34+ cells during transduction expanded a subpopulation of CD14+/CD31+ monocytes with excellent ability to carry the FVIII transgene, allowing the correction of HA phenotype in zebrafish. Finally, the HA zebrafish model showed that f8 RNA is predominantly localized in the hematopoietic system at the larval stage, which indicates a potential contributory role of FVIII in hematopoiesis that warrants further investigation. We believe that this study may be of broad interest to hematologists and researchers striving to advance knowledge and permanent treatments for patients with HA.

Poster by Houda Yasmine Ali Moussa (Hamad Bin Khalifa University) , Nimshitha Manaph (Hamad Bin Khalifa University), Gowher Ali (Hamad Bin Khalifa University), Selma Maacha (Sidra Medicine), Kyung Chul Shin (Hamad Bin Khalifa University),, Samia M. Ltaief (Hamad Bin Khalifa University), Vijay Gupta (Hamad Bin Khalifa University), Yongfeng Tong (Hamad Bin Khalifa University), Janarthanan Ponraj (Hamad Bin Khalifa University), Salam Salloum-Asfar (Hamad Bin Khalifa University), Said Mansour (Hamad Bin Khalifa University), Fouad A. Al-Shaban (Hamad Bin Khalifa University), Hyung-Goo Kim (Hamad Bin Khalifa University), Lawrence W Stanton (Hamad Bin Khalifa University), Jean-Charles Grivel (Sidra Medicine), Sara A Abdulla (Hamad Bin Khalifa University), Abeer R. Al-Shammari (Hamad Bin Khalifa University), Yongsoo Park (Hamad Bin Khalifa University) Background: Extracellular vesicles (EVs) are membrane vesicles released from cells to the extracellular space, involved in cell-to-cell communication by the horizontal transfer of biomolecules such as proteins and RNA. Because EVs can cross the blood-brain barrier (BBB), circulating through the bloodstream and reflecting the cell of origin in terms of disease prognosis and severity, the contents of plasma EVs provide non-invasive biomarkers for neurological disorders. However, neuronal EV markers in blood plasma remain unclear. EVs are very heterogeneous in size and contents, thus bulk analyses of heterogeneous plasma EVs using Western blot and ELISA have limited utility. Objective: We aim to identify specific markers for brain-derived EVs isolated from plasma for biomarkers of neuronal disorders. Methods: Using flow cytometry to analyze individual neuronal EVs, we show that our plasma EVs isolated by size exclusion chromatography are mainly CD63-positive exosomes of endosomal origin. We have also used human induced pluripotent stem cells (iPSCs)-derived cortical neurons and brain organoids to study neuronal EVs. Results: Neural cell adhesion molecule (NCAM) is highly expressed and enriched in EVs released from human iPSCs-derived cortical neurons and organoids. Subpopulations of blood plasma exosomes contain NCAM, suggesting its origin from neuronal cells. We identified the subpopulations of plasma EVs that contain NCAM using flow cytometry-based individual EV analysis. Conclusion: Our data propose that NCAM-positive neuronal exosomes isolated from blood plasma can be detected and analyzed using flow cytometry. Neuronal EVs identified here will pave the way for further studies to discover biomarkers for neurological disorders.

Fanconi–Bickel Syndrome (FBS) is a rare disorder of carbohydrate metabolism that is characterized by the accumulation of glycogen mainly in the liver. It is inherited in an autosomal recessive manner due to mutations in the SLC2A2 gene. SLC2A2 encodes for the glucose transporter GLUT2 and is expressed in tissues that are involved in glucose homeostasis. The molecular mechanisms of dysglycemia in FBS are still not clearly understood. In this study, we report two cases of FBS with classical phenotypes of FBS associated with dysglycemia. Genomic DNA was extracted and analyzed by whole-genome and Sanger sequencing, and patient PBMCs were used for molecular analysis. One patient had an exonic SLC2A2 mutation (c.1093C > T in exon 9, R365X), while the other patient had a novel intronic SLC2A2 mutation (c.613-7T>G). Surprisingly, the exonic mutation resulted in the overexpression of dysfunctional GLUT2, resulting in the dysregulated expression of other glucose transporters. The intronic mutation did not affect the coding sequence of GLUT2, its expression, or glucose transport activity. However, it was associated with the expression of miRNAs correlated with type 1 diabetes mellitus, with a particular significant overexpression of hsa-miR-29a-3p implicated in insulin production and secretion. Our findings suggest that SLC2A2 mutations cause dysglycemia in FBS either by a direct effect on GLUT2 expression and/or activity or, indirectly, by the dysregulated expression of miRNAs implicated in glucose homeostasis.

Extracellular vesicles (EVs) are membrane vesicles released from cells to the extracellular space, involved in cell-to-cell communication by the horizontal transfer of biomolecules such as proteins and RNA. Because EVs can cross the blood-brain barrier (BBB), circulating through the bloodstream and reflecting the cell of origin in terms of disease prognosis and severity, the contents of plasma EVs provide non-invasive biomarkers for neurological disorders. However, neuronal EV markers in blood plasma remain unclear. EVs are very heterogeneous in size and contents, thus bulk analyses of heterogeneous plasma EVs using Western blot and ELISA have limited utility. In this study, using flow cytometry to analyze individual neuronal EVs, we show that our plasma EVs isolated by size exclusion chromatography are mainly CD63-positive exosomes of endosomal origin. As a neuronal EV marker, neural cell adhesion molecule (NCAM) is highly enriched in EVs released from induced pluripotent stem cells (iPSCs)-derived cortical neurons and brain organoids. We identified the subpopulations of plasma EVs that contain NCAM using flow cytometry-based individual EV analysis. Our results suggest that plasma NCAM-positive neuronal EVs can be used to discover biomarkers for neurological disorders.

Fanconi–Bickel Syndrome (FBS) is a rare disorder of carbohydrate metabolism that is characterized mainly by the accumulation of glycogen in the liver and kidney. It is inherited as an autosomal recessive disorder caused by mutations in the SLC2A2 gene, which encodes for GLUT2. Patients with FBS have dysglycemia but the molecular mechanisms of dysglycemia are still not clearly understood. Therefore, we aimed to understand the underlying molecular mechanisms of dysglycemia in a patient with FBS. Genomic DNA was isolated from a peripheral blood sample and analyzed by whole genome and Sanger sequencing. CRISPR-Cas9 was used to introduce a mutation that mimics the patient’s mutation in a human kidney cell line expressing GLUT2 (HEK293T). Mutant cells were used for molecular analysis to investigate the effects of the mutation on the expression and function of GLUT2, as well as the expression of other genes implicated in dysglycemia. The patient was found to have a homozygous nonsense mutation (c.901C>T, R301X) in the SLC2A2 gene. CRISPR-Cas9 successfully mimicked the patient’s mutation in HEK293T cells. The mutant cells showed overexpression of a dysfunctional GLUT2 protein, resulting in reduced glucose release activity and enhanced intracellular glucose accumulation. In addition, other glucose transporters (SGLT1 and SGLT2 in the kidney) were found to be induced in the mutant cells. These findings suggest the last loops (loops 9-12) of GLUT2 are essential for glucose transport activity and indicate that GLUT2 dysfunction is associated with dysglycemia in FBS.

Detection of factor VIII (FVIII) in cells by flow cytometry is controversial, and no monoclonal fluorescent antibody is commercially available. In this study, we optimized such an assay and successfully used it as a platform to study the functional properties of phosphoglycerate kinase (PGK)-FVIII lentiviral vector-transduced cells by directly visualizing FVIII in cells after different gene transfer conditions. We could measure cellular stress parameters after transduction by correlating gene expression and protein accumulation data. Flow cytometry performed on transduced cell lines showed that increasing MOI rates resulted in increased protein levels, plateauing after an MOI of 30. We speculated that, at higher MOI, FVIII production could be impaired by a limiting factor required for proper folding. To test this hypothesis, we interfered with the unfolded protein response by blocking proteasomal degradation and measured the accumulation of intracellular misfolded protein. Interestingly, at higher MOIs the cells displayed signs of toxicity with reactive oxygen species accumulation. This suggests the need for identifying a safe window of transduction dose to avoid consequent cell toxicity. Herein, we show that our flow cytometry platform for intracytoplasmic FVIII protein detection is a reliable method for optimizing gene therapy protocols in hemophilia A by shedding light on the functional status of cells after gene transfer., Graphical Abstract

Two-dimensional transition metal carbides/carbonitrides known as MXenes are rapidly growing as multimodal nanoplatforms in biomedicine. Here, taking SARS-CoV-2 as a model, we explored the antiviral properties and immune-profile of a large panel of four highly stable and well-characterized MXenes - Ti3C2Tx, Ta4C3Tx, Mo2Ti2C3Tx and Nb4C3Tx. To start with antiviral assessment, we first selected and deeply analyzed four different SARS-CoV-2 genotypes, common in most countries and carrying the wild type or mutated spike protein. When inhibition of the viral infection was tested in vitro with four viral clades, Ti3C2Tx in particular, was able to significantly reduce infection only in SARS-CoV-2/clade GR infected Vero E6 cells. This difference in the antiviral activity, among the four viral particles tested, highlights the importance of considering the viral genotypes and mutations while testing antiviral activity of potential drugs and nanomaterials. Among the other MXenes tested, Mo2Ti2C3Tx also showed antiviral properties. Proteomic, functional annotation analysis and comparison to the already published SARS-CoV-2 protein interaction map revealed that MXene-treatment exerts specific inhibitory mechanisms. Envisaging future antiviral MXene-based drug nano-formulations and considering the central importance of the immune response to viral infections, the immune impact of MXenes was evaluated on human primary immune cells by flow cytometry and single-cell mass cytometry on 17 distinct immune subpopulations. Moreover, 40 secreted cytokines were analyzed by Luminex technology. MXene immune profiling revealed i) the excellent bio and immune compatibility of the material, as well as the ability of MXene ii) to inhibit monocytes and iii) to reduce the release of pro-inflammatory cytokines, suggesting an anti-inflammatory effect elicited by MXene. We here report a selection of MXenes and viral SARS-CoV-2 genotypes/mutations, a series of the computational, structural and molecular data depicting deeply the SARS-CoV-2 mechanism of inhibition, as well as high dimensional single-cell immune-MXene profiling. Taken together, our results provide a compendium of knowledge for new developments of MXene-based multi-functioning nanosystems as antivirals and immune-modulators., Graphical abstract

Comme il y a un secret de Monte-Cristo, il y a un secret d’Alexandre Dumas. Ce secret concerne l’identité de l’écrivain, et non plus les motifs qui ont dicté le choix du nom porté par le héros. Chacun de ses écrits, de façon éminemment cryptée, remonte à une origine « nègre » rebelle - sa grand’mère était esclave -, mais fait appel aussi à une origine « blanche » conforme - son grand-père était un hobereau normand parti chercher fortune aux Îles dans le sucre et la traite -, qu’il a décidé toutes deux de revendiquer. Le présent ouvrage explore les méandres suivis à travers l’œuvre dans ce difficile jeu de bascule et met au jour ce qu’il en est de la véritable négritude d’âme et de peau assumée par Dumas. Il montre quels curieux substituts certains animaux totémiques offrent au fervent chasseur que celui-ci affiche être. Il découvre aussi par quelle « cuisine » la bête élue doit être apprêtée pour convenir au destin - au festin - symbolique qui est, de toute nécessité, le sien. Ce n’est donc pas pour rien que l’écrivain clot son œuvre par un Dictionnaire de Cuisine. Une page est un plat, et la viande esprit, si l’encre est farine.

Esophageal cancer (EC) is a disease often marked by aggressive growth and poor prognosis. Lack of targeted therapies, resistance to chemoradiation therapy, and distant metastases among patients with advanced disease account for the high mortality rate. The tumor microenvironment (TME) contains several cell types, including fibroblasts, immune cells, adipocytes, stromal proteins, and growth factors, which play a significant role in supporting the growth and aggressive behavior of cancer cells. The complex and dynamic interactions of the secreted cytokines, chemokines, growth factors, and their receptors mediate chronic inflammation and immunosuppressive TME favoring tumor progression, metastasis, and decreased response to therapy. The molecular changes in the TME are used as biological markers for diagnosis, prognosis, and response to treatment in patients. This review highlighted the novel insights into the understanding and functional impact of deregulated cytokines and chemokines in imparting aggressive EC, stressing the nature and therapeutic consequences of the cytokine-chemokine network. We also discuss cytokine-chemokine oncogenic potential by contributing to the Epithelial-Mesenchymal Transition (EMT), angiogenesis, immunosuppression, metastatic niche, and therapeutic resistance development. In addition, it discusses the wide range of changes and intracellular signaling pathways that occur in the TME. Overall, this is a relatively unexplored field that could provide crucial insights into tumor immunology and encourage the effective application of modulatory cytokine-chemokine therapy to EC.

Blood doping in sports is prohibited by the World Anti-Doping Agency (WADA). To find a possible biomarker for the detection of blood doping, we investigated the changes in blood stored in CPDA-1 blood bags of eight healthy subjects who donated one unit of blood. Aliquots were taken on days 0, 14, and 35. Platelet-free plasma was prepared and stored at -80°C until analysis on a flow cytometer dedicated for the analysis of microparticles (MPs). Changes in the number of red blood cell (RBC) -MPs were highly significant (p < 0.0001) with a mean of 219 (10^3/μL) on day 0 changing to 23 120 (10^3/μL) on day 14 and 29 310 (10^3/ μL) on day 35. We conclude that RBC-MPs seem to be a promising biomarker for doping control but confirmation by a transfusion study is necessary.

Introduction UM171, a novel compound with hematopoietic stem cells (HPSCs) self-renewal properties has shown great potential for expanding CD34+ HPSCs while preserving their stemness and increasing their engrafting abilities. The compound has been increasingly used for the purpose of CD34+ HPSCs expansion, both in pre-clinical research and in clinical applications. Its use is particularly important in the gene therapy (GT) and bone marrow transplantation (BMT) fields, especially when CD34+ expansion is needed to ensure the success of the therapy. Recent reports have demonstrated UM171efficacy to expand enriched CD34+ HPSCs from peripheral blood (PB) in osteopetrosis patients, and after mobilization in lymphoma patients. Here, we demonstrate the feasibility of exploiting the UM171 molecule to directly expand CD34+ HPSCs in bulk PB mononuclear cells (MNCs) without the need of either HPSCs mobilization or CD34+ enrichment. Material and Methods Our protocol entails culturing of bulk MNCs directly after Ficoll gradient separation in IMDM media supplemented with 10% FBS, and HPSCs stemness sparing cytokines, used in clinical suitable concentrations: SCF 300 ng/ml, TPO 100 ng/ml, Flt3-L 300 ng/ml and IL3 60 ng/ml +/- 38 nM UM171 for 20 days. Briefly, quantities of 7.5± 0.5 ml of blood from 2 healthy donors were gradient-separated by Ficoll and yielded a total of 6.6±0.2X10^6 MNCs. Cells were divided in two conditions/donor (Cytokines +/- UM171) and 3.3±0.14X10^6 cell were seeded in 6 well plates at concentration of 1.1X10^6/ml in a total volume of 3 ml. Half of medium was changed every 5 days. Cell count and phenotype of HPSCs were periodically evaluated to determine the optimal expansion window. Cells were manually counted, and phenotype was measured through Symphony A5 flow-cytometry with this antibody panel: Zombie viability dye, CD45, CD34, and in the last timepoint (day 20) CD11b as an exclusion marker for myeloid differentiation. Results UM171 treatment efficiently achieved direct expansion of CD34+ HPSCs in bulk PB MNCs, reaching a peak expansion at day 12 with CD34+ Cells representing 3.9±0.3% of cells (mean±SD of live cells) compared to 0.08±0.04% at baseline (48.75x increase). This increase in CD34+ cell concentration was also partially due to the decline of total PB MNCs cell count during culture time (declined up to 90%). Despite the overall cell count decrease, the CD34+ HPSCs population had an absolute number increase of 6.5±0.916 times with respect to the initial count, peaking at day 12 with an absolute number of 19600±1344 cells compared to 2985±1466 cells at baseline. If projected to a larger scale, this yield proves the feasibility of obtaining of ∼20X10^6 CD34+ HPSCs from a buffy coat of 350 ml blood donation. This amount would be adequate for a pediatric HSCT, and very handy for multiple experiments with CD34+ HPSCs. Conclusion In conclusion, we have provided a proof of concept on the feasibility to directly expand CD34+ HPSCs in bulk PB MNCs. Further characterization of the expanded CD34+ will be needed to ensure stemness, plasticity, and engrafting ability. The advantage of expanding HPSCs in bulk is to preserve the rare naïve CD34+ population that can be lost during the immunoselection process and to take advantage of the myeloid niche, to further enhance the expansion along with UM171. This expansion approach can be performed at larger scale on the buffy coats often dismissed in blood banks to provide considerable number of CD34+ cells for either banking, research or future clinical application. Disclosures No relevant conflicts of interest to declare.

Introduction Acute Lymphoblastic Leukemia (ALL) is the most common malignancy in children and represents 75-80% of leukemia cases. Despite its recognized role in the initiation of the disease, the bone marrow (BM) microenvironment is not well understood especially in its non-cellular component. High-throughput protein detection represent an opportunity to unveil new biomarkers for leukemia diagnosis, prognosis, monitoring, and to potentially identify biological targets. Methods In the present exploratory study, we evaluated the soluble compartment of BM and peripheral blood (PB) in 16 ALL pediatric patients by the means of the SOMAscan assay, a highly multiplexed, affinity proteomics platform able to measure 1305 proteins in as little as 65µl of sample using modified protein-binding single-stranded DNA aptamers called SOMAmers, revealed on a high dimension oligo-probe array. The raw hybridization data were normalized using hybridization controls and were log2-transformed. Transformed HybNormalized BM and PB plasma data were compared using GraphPad PRISM 8. Multiple comparisons were set with an FDR threshold of 0.01%. Results The comparative analysis between plasma samples from BM and PB showed that 228 proteins are differentially expressed in BM and PB plasma irrespective of the nature of disease (fig1). A more detailed mix-effect analysis of protein expression in the different patient categories and compartment defined in Table 1 revealed that out of these 228 proteins, 92 showed a significant differential expression when performing a mixed-effect analysis between the different groups. Among the top 20 proteins differentially expressed between BM and PB, 18 show differential expression between patient groups. The patient group effect is dominated by the differences between BM and PB plasma in ALL Common patients , which account for 17 of the observed differences in protein levels. In spite of the limited sample size, the analysis revealed a difference in NOTC2 level between ALL Common PB and ALL Pro-B PB. Conclusion In our study we applied a quantitative large-scale proteomic analysis on BM and PB plasma in children suffering from ALL, identifying differential protein expression between the two compartments. In the last few years, SomaScan has emerged as a very attractive method due to its high throughput capacity, faster discovery mode compared to other proteomic platforms and small sample size required, which make it ideal for the identification of novel biomarkers. In spite of the limiting samples sizes, some differences in protein expression were revealed. We are taking this analysis further by analyzing more patients in different groups and including normal donor samples. Table 1 Table Disclosures No relevant conflicts of interest to declare.

The role of the mesenchymal stromal cell- (MSC-) derived secretome is becoming increasingly intriguing from a clinical perspective due to its ability to stimulate endogenous tissue repair processes as well as its effective regulation of the immune system, mimicking the therapeutic effects produced by the MSCs. The secretome is a composite product secreted by MSC in vitro (in conditioned medium) and in vivo (in the extracellular milieu), consisting of a protein soluble fraction (mostly growth factors and cytokines) and a vesicular component, extracellular vesicles (EVs), which transfer proteins, lipids, and genetic material. MSC-derived secretome differs based on the tissue from which the MSCs are isolated and under specific conditions (e.g., preconditioning or priming) suggesting that clinical applications should be tailored by choosing the tissue of origin and a priming regimen to specifically correct a given pathology. MSC-derived secretome mediates beneficial angiogenic effects in a variety of tissue injury-related diseases. This supports the current effort to develop cell-free therapeutic products that bring both clinical benefits (reduced immunogenicity, persistence in vivo, and no genotoxicity associated with long-term cell cultures) and manufacturing advantages (reduced costs, availability of large quantities of off-the-shelf products, and lower regulatory burden). In the present review, we aim to give a comprehensive picture of the numerous components of the secretome produced by MSCs derived from the most common tissue sources for clinical use (e.g., AT, BM, and CB). We focus on the factors involved in the complex regulation of angiogenic processes.

Background An association between productive cytomegalovirus infection and atherosclerosis was shown recently in several trials, including a previous study of ours. However, the mechanism involved in this association is still under investigation. Here, we addressed the interaction between productive cytomegalovirus infection and endothelial function in patients with ST-elevation myocardial infarction (STEMI). Methods We analyzed the presence of cytomegaloviral DNA in plasma and endothelial function in 33 patients with STEMI and 33 volunteers without cardiovascular diseases, using real-time polymerase chain reaction (PCR) and a noninvasive test of flow-mediated dilation. Results Both the frequency of presence and the load of cytomegaloviral DNA were higher in plasma of patients with STEMI than those in controls. This difference was independent of other cardiovascular risk factors (7.38 [1.36-40.07]; P = 0.02). The results of the flow-mediated dilation test were lower in patients in STEMI than in controls (5.0% [2.65%-3.09%] vs 12. %5 [7.5%-15.15%]; P = 0.004) and correlated negatively with the cytomegaloviral DNA load (Spearman R = −0.407; P = 0.019) independently of other cardiovascular risk factors. Conclusions Productive cytomegalovirus infection in patients with STEMI correlated negatively with endothelial function independently of other cardiovascular risk factors. The impact of cytomegalovirus on endothelial function may explain the role of cytomegalovirus in cardiovascular prognosis.

The tumor microenvironment represents a complex network, in which tumor cells not only communicate with each other but also with stromal and immune cells. Current research has demonstrated the vital role of the tumor microenvironment in supporting tumor phenotype via a sophisticated system of intercellular communication through direct cell-to-cell contact or by classical paracrine signaling loops of cytokines or growth factors. Recently, extracellular vesicles have emerged as an important mechanism of cellular interchange of bioactive molecules. Extracellular vesicles isolated from tumor and stromal cells have been implicated in various steps of tumor progression, such as proliferation, angiogenesis, metastasis, and drug resistance. Inhibition of extracellular vesicles secretion, and thus of the transfer of oncogenic molecules, holds promise for preventing tumor growth and drug resistance. This review focuses on the role of extracellular vesicles in modulating the tumor microenvironment by addressing different aspects of the bidirectional interactions among tumor and tumor-associated cells. The contribution of extracellular vesicles to drug resistance will also be discussed as well as therapeutic strategies targeting extracellular vesicles production for the treatment of cancer.

Since the publication of the Society for Immunotherapy of Cancer’s (SITC) original cancer immunotherapy biomarkers resource document, there have been remarkable breakthroughs in cancer immunotherapy, in particular the development and approval of immune checkpoint inhibitors, engineered cellular therapies, and tumor vaccines to unleash antitumor immune activity. The most notable feature of these breakthroughs is the achievement of durable clinical responses in some patients, enabling long-term survival. These durable responses have been noted in tumor types that were not previously considered immunotherapy-sensitive, suggesting that all patients with cancer may have the potential to benefit from immunotherapy. However, a persistent challenge in the field is the fact that only a minority of patients respond to immunotherapy, especially those therapies that rely on endogenous immune activation such as checkpoint inhibitors and vaccination due to the complex and heterogeneous immune escape mechanisms which can develop in each patient. Therefore, the development of robust biomarkers for each immunotherapy strategy, enabling rational patient selection and the design of precise combination therapies, is key for the continued success and improvement of immunotherapy. In this document, we summarize and update established biomarkers, guidelines, and regulatory considerations for clinical immune biomarker development, discuss well-known and novel technologies for biomarker discovery and validation, and provide tools and resources that can be used by the biomarker research community to facilitate the continued development of immuno-oncology and aid in the goal of durable responses in all patients.

The relationship between acute coronary syndrome (ACS) and local and systemic inflammation, including accumulation of macrophages in atherosclerotic plaques and upregulation of blood cytokines (e.g., C-reactive protein (CRP)), has been known for more than 100 years. The atherosclerosis-associated inflammatory response has been traditionally considered as an immune system reaction to low-density lipoproteins. At the same time, some data have indicated a potential involvement of cytomegalovirus (CMV) in the activation and progression of atherosclerosis-associated inflammation, leading to ACS. However, these data have been tangential and mainly concerned the relationship between a coronary artery disease (CAD) prognosis and the anti-CMV antibody titer. We assumed that ACS might be associated with CMV reactivation and virus release into the bloodstream. The studys aim was to test this assumption through a comparison of the plasma CMV DNA level in patients with various CAD forms and in healthy subjects. To our knowledge, no similar research has been undertaken yet. A total of 150 subjects (97 CAD patients and 53 healthy subjects) were examined. Real-time polymerase chain reaction (RT-PCR) was used to determine the number of plasma CMV DNA copies. We demonstrated that the number of plasma CMV genome copies in ACS patients was significantly higher than that in healthy subjects (p = 0.01). The CMV genome copy number was correlated with the plasma CRP level (p = 0.002). These findings indicate a potential relationship between CMV activation and atherosclerosis exacerbation that, in turn, leads to the development of unstable angina and acute myocardial infarction. Monitoring of the CMV plasma level in CAD patients may be helpful in the development of new therapeutic approaches to coronary atherosclerosis treatment.

Cytotoxic T cells (CTLs) and B cells engage distinct interactions in GVHD patients' blood and tissues, detectable in regular flow-cytometry screenings, by size and by double positive CD19-CD8 antibody markers (Deola, BMT 2017). B-CTL couplets are formed by alpha-betaTCR+ CD8+ CTLs preferentially targeting CD27+ CD19+ cells displaying an activated CD80 and CD86 phenotype. Interactions may last from 5 minutes to roughly 1 hour, and release a pattern of T cell attracting chemokines, as IP10, MIG, ITAC, which are also known GVHD biomarkers. To further unravel the mechanism of this cell interaction, we built an in-vitro model where human PBMCs cells are expanded with cognate peptides and IL2 for 1-2 weeks, then immune-selected for CD8 antigen by Miltenyi microbeads negative-selection and incubated (2-18 hours) with fresh autologous CD19-B cells, immune-selected with the same method. The interactions are studied under confocal microscope video-imaging (Zeiss LSM 880+Imaris 3D analysis software) and in flow-cytometry (SymphonyA5 BD) after deep phenotype antibody staining. The intensity of interaction, measured by fluorescence interference on cell membranes, revealed an active engagement of CD19 and CD8 antigens. CD19 antigen penetrates deeper in contacting T cells, than CD8 on B cells, and consistently with this finding, after the interactions there is an antigen exchange between cells with CD19 antigen actively transferred in CD8 cells (p value = We already proved that this type of B-T interaction is not antigen specific in CTL-to-B direction (Deola et a, JI 2008) but to exclude cross-presentation from B to CTLs and to unravel the role of CD8, we interfered by antibody blocking of MHC class I pathway on B cells and CD8 on CTLs. B-T cell interactions are not abolished after MHC-I or CD8 blocking, the intensity of coupling is unchanged after MHC-I block, and is higher after blocking CD8 (p value= Interestingly, B cell engagement follows 2 repetitive patterns of interaction: a high intensity interaction that visually corresponds to tight coupling cells with high CD19 penetration in T cells, and a low-intensity continuous interaction, visually measurable by cells "sniffing" each other. Both patterns correspond to diverse Calcium flux activation on T cells and B cells, suggesting functional different pathways triggered by the 2 type of interactions. Deep phenotype flow cytometry analyses after coupling reveals distinct programs triggered by the contact in both B cells and T cells. While after the interaction CTLs double their pool of perforin bearing effectors and their fraction of CD45RA-/CD27+ memory CTLs, CD19 preferentially undergo a deletion of IgD- CD27- (DN) cells (13,85%+/-1,1 and 22,95%+/-4,5 CD95/Fas+, respectively in B cells alone and B+CTLs, n=2) and a rescue of affinity mature CD27+ IgD- cells (39.8%+/-25,47 and 21,2%+/- 29% CD95/Fas+ in the same groups) CTLs are the ultimate line of "tissue attack" in GVHD and several diseases, as autoimmune diseases, cancer, viral diseases, sharing a common pathological program definable as "immune rejection". B cells are key players in immune rejection, but a link between these 2 types of cells is still unclear. Our findings enforce the hypothesis of a program of peripheral tolerance/activation triggered directly between B cells and activated CTLs in the context of inflammation and of GVHD. Disclosures No relevant conflicts of interest to declare.

Textvles - Le Fantastique peut-il etre compris comme une question de regard, de point de vue ? Charles Grivel - Je le dis dans mon livre, je le crois. Un œil se rencontre lui-meme dans les emanations qu’il execute. Il s’y plait, il s’y deplait aussi. Il y reconnait. Il en redemande. Vous dites « perspective » ? Moins. L’œil ne se definit pas par la perspective : il est large, trop large, il aimerait un peu plus des œilleres : la « perspective » est une invention de narratologue. Pour moi, les...

Extracellular vesicles (EVs) are important in normal physiology and are altered in various pathologies. EVs produced by different cells are antigenically different. Since the majority of EVs are too small for routine flow cytometry, EV composition is studied predominantly in bulk, thus not addressing their antigenic heterogeneity. Here, we describe a nanoparticle-based technique for analyzing antigens on single nano-sized EVs. The technique consists of immuno-capturing of EVs with 15-nm magnetic nanoparticles, staining captured EVs with antibodies against their antigens, and separating them from unbound EVs and free antibodies in a magnetic field, followed by flow analysis. This technique allows us to characterize EVs populations according to their antigenic distribution, including minor EV fractions. We demonstrated that the individual blood EVs carry different sets of antigens, none being ubiquitous, and quantified their distribution. The physiological significance of antigenically different EVs and their correlation with different pathologies can now be directly addressed.From the Clinical EditorThis study reports a nanoparticle-based technique for analyzing antigens on single nano-sized extracellular vehicles (EV). The technique consists of immuno-capturing of EVs with 15-nm magnetic nanoparticles, followed by staining the captured EVs with antibodies and separating them via a magnetic field, followed by flow analysis. This technique enables studies of antigenic properties of individual EVs that conventionally can only be studied in bulk.

Problem To characterize the amniotic fluid (AF) inflammatory-related protein (IRP) network in patients with a sonographic short cervix (SCx) and to determine its relation to early preterm delivery (ePTD). Method of study A retrospective cohort study included women with a SCx (≤25 mm; n=223) who had amniocentesis and were classified according to gestational age (GA) at diagnosis and delivery (ePTD

HIV-1 envelope proteins (Envs) play a critical role in HIV infection. In a correct trimeric conformation, Env mediates virus–cell binding and fusion. Malfunctioning of this machinery renders virions incapable of infecting cells. Each HIV-1 virion carries 10–14 Envs, and therefore a defective Env may not necessarily render a HIV virion non-infectious, since other Env on the same virion may still be functional. Alternatively, it is possible that on a given virion either all the spikes are defective or all are functional. Here, we investigate Env conformations on individual virions using our new nanotechnology, “flow virometry”, and a panel of antibodies that discriminate between various Env conformations. We found that the majority of HIV-1 virions carry either only trimeric (“functional”) or only defective spikes. The relatively small subfraction of virions that carry both functional and nonfunctional Envs contributes little to HIV infection of human lymphoid tissue ex vivo. The observation that the majority of virions exclusively express either functional or nonfunctional forms of Env has important implications for understanding the role of neutralizing and non-neutralizing antibodies in the immune control of HIV infection as well as for the development of effective prophylactic strategies.

Cells release small extracellular vesicles (EVs) into the surrounding media. Upon virus infection cells also release virions that have the same size of some of the EVs. Both virions and EVs carry proteins of the cells that generated them and are antigenically heterogeneous. In spite of their diversity, both viruses and EVs were characterized predominantly by bulk analysis. Here, we describe an original nanotechnology-based high throughput method that allows the characterization of antigens on individual small particles using regular flow cytometers. Viruses or extracellular vesicles were immunocaptured with 15 nm magnetic nanoparticles (MNPs) coupled to antibodies recognizing one of the surface antigens. The captured virions or vesicles were incubated with fluorescent antibodies against other surface antigens. The resultant complexes were separated on magnetic columns from unbound antibodies and analyzed with conventional flow cytometers triggered on fluorescence. This method has wide applications and can be used to characterize the antigenic composition of any viral- and non-viral small particles generated by cells in vivo and in vitro. Here, we provide examples of the usage of this method to evaluate the distribution of host cell markers on individual HIV-1 particles, to study the maturation of individual Dengue virions (DENV), and to investigate extracellular vesicles released into the bloodstream.

Cancer immunotherapies are showing promising clinical results in a variety of malignancies. Monitoring the immune as well as the tumor response following these therapies has led to significant advancements in the field. Moreover, the identification and assessment of both predictive and prognostic biomarkers has become a key component to advancing these therapies. Thus, it is critical to develop systematic approaches to monitor the immune response and to interpret the data obtained from these assays. In order to address these issues and make recommendations to the field, the Society for Immunotherapy of Cancer reconvened the Immune Biomarkers Task Force. As a part of this Task Force, Working Group 3 (WG3) consisting of multidisciplinary experts from industry, academia, and government focused on the systematic assessment of immune regulation and modulation. In this review, the tumor microenvironment, microbiome, bone marrow, and adoptively transferred T cells will be used as examples to discuss the type and timing of sample collection. In addition, potential types of measurements, assays, and analyses will be discussed for each sample. Specifically, these recommendations will focus on the unique collection and assay requirements for the analysis of various samples as well as the high-throughput assays to evaluate potential biomarkers.

INTRODUCTION Mesenchymal Stromal Cells (MSCs) are multipotent cells with regenerative, anti-inflammatory, immunomodulatory and anti-tumorigenic properties, which are readily isolated from aspirates of adipose tissue (AT) due to their high availability. Compared with alternative sources, AT provides high cell yield. Despite the numerous clinical trials in autologous and allogeneic settings for various diseases worldwide, there are no US FDA-approved MSC-based products. Challenges for clinical translation of MSC-based therapies largely lie in the sourcing, production and basic characterization of such products (Camilleri et al, 2016; Mendicino et al, 2014). Given the known donor-related variability (sex, age, liposuction sites, BMI, etc.), there is still ample room to: better standardize in vitro manufacturing processesrefine the characterization of clinical products. METHODS Seven samples of lipoaspirate were processed. AT manual digestion was compared with an automated collagenase-based enzymatic digestion by the Cytori Celution instrument, a closed system regulated as a medical device by EC for the production of adipose-derived stem cells-enriched products. Total cell yield, cell harvest at P0, P1 and P2, Population Doubling Level (PDL) and Doubling Time (DT) were evaluated. The obtained Adipose-derived Stromal Cells (ADSCs) were characterized for: Expression of 361 surface markers (BioLegend LegendScreen Human PE Kit), including 117 additional antigens compared to other reported screens. Cytori-isolated ADSCs from three donors for 6 total conditions (3 P2, 2 P3 and 1 IFN-g-primed P2) were screened. A broader characterization of fresh isolates (P0) as well as (P3) from the same donors was performed with a 20-color flow cytometric panel. Data were analyzed with FlowJo software.miRNA expression was evaluated using the 800 human miRNAs Nanostring panel. Nanostring nCounter Analysis System generated data, then Partek GS software was used for secondary analysis. RESULTS Cytori processing resulted in a higher cell yield at isolation (p= 0.0156), and ADSCs exhibited higher proliferation in vitro at P0 (n. of days at P0, p=0.0313), higher cell yield at P1 (p=0.0313), and higher PDL1 (p=0.0469). Out of the 361 markers examined, in every donor at each passage, at least 94 were found positive (expression >1%), including 30 within the newly screened 117 markers. Altogether, 27 markers were expressed at more than 85%, including 6 from the newly screened markers. Our analysis identified 50 donor-dependent markers, 50 passage-modulated markers (P2 vs P3) and 45 IFN-g-modulated marker, 41 increased and 4 decreased. We were able to identify the ADSCs progenitors markers CD146 and CD271 on cell as in P0 and P3, we observed a transition from a dominant expression in P0 of CD271 (34.05%) vs CD146 (1.62% ) to a dominant expression of CD146 in P3 (52%) vs CD271 (0.7%) in a single donor. The frequency of double positives CD146+CD271+ was 1% and 0.6% in P0 and P3 respectively. miRNA expression analysis by ANOVA revealed that (i) IFN-g-priming modulated 19 miRNAs, that 4 miRNAs were modulated by passage number and donors' origin. CONCLUSION Our study shows that the standardized Cytori processing advantageously substitutes AT manual digestion by enabling higher cell yield and potentially providing multiple ADSC doses from a single donor. The isolation procedure is standardized, the operator-dependent variations are minimized, and less prone to contamination compared to the lengthy, multistep process of manual digestion. The broad cell characterization with flow cytometry and miRNA expression revealed the expression and modulation of new markers. We believe that increasing the dimensionality of the ADSC characterization, beyond the traditional markers, could reveal markers that describe specific functional abilities of clinical ADSC product. Proper functional studies are necessary to validate our hypothesis. Disclosures No relevant conflicts of interest to declare.

"Cervical tissue explants (CTEs) from 22 HIV-1 seronegative women were exposed to . R5 HIV-1 ex vivo. Eight CTEs were productively infected in terms of HIV-1 p24Gag . release in culture supernatants, whereas 14 were not. Nonetheless, both. accumulation of HIV-1gag DNA and of p24Gag(+) CD4(+) T cells and macrophages. occurred in both productive and, at lower levels, in nonproductive CTEs.. Nonproductive CTEs differed from productive CTEs for higher secretion of C-C. motif chemokine ligand 3 (CCL3) and CCL5. A post-hoc analysis revealed that all. productive CTEs were established from women in their secretory phase of the. menstrual cycle, whereas nonproductive CTEs were derived from women either in. their secretory (28%) or proliferative (36%) menstrual cycle phases or with an. atrophic endometrium (36%). Thus, our results support the epidemiological. observation that sexual HIV-1 transmission from males to women as well as from. women to men is more efficient during their secretory phase of the menstrual. cycle."

Background Although an association between human herpesvirus ( HHV ) infection and atherosclerosis has been suggested, the data supporting such an association are controversial and, in most cases, are based on serological evidence or on the presence of cell‐associated HHV DNA , which do not report about actual viral replication. We quantified the DNA of all 8 types of HHVs in plasma, in which their presence is evidence of viral replication. Methods and Results Using quantitative real‐time polymerase chain reaction, we evaluated the presence of HHV DNA in blood samples obtained at the time of hospitalization from 71 patients with acute coronary syndrome, 26 patients with stable coronary artery disease, and 53 healthy volunteers and in atherosclerotic plaques of 22 patients with peripheral artery disease who underwent endarterectomy. HHV ‐5 (cytomegalovirus [ CMV ]) was the only HHV with a level that was higher in acute coronary syndrome patients than in the control group and that correlated with the level of high‐sensitivity C‐reactive protein. The numbers of effector memory T cells positively correlated with the numbers of CMV genome copies in carotid arteries plaques, whereas the numbers of central memory T cells negatively correlated with CMV copy numbers. Conclusions Of all HHV levels, only CMV was higher in patients with stable coronary artery disease and acute coronary syndrome than in the healthy group, and its load correlated with the level of high‐sensitivity C‐reactive protein. The level of CMV in atherosclerotic plaques correlated with the state of immunoactivation of lymphocytes in plaques, suggesting that the reactivation of CMV may contribute to the immune activation associated with the progression of atherosclerosis.