Vaginal neutrophils eliminate sperm by trogocytosis

2020 ◽  
Vol 35 (11) ◽  
pp. 2567-2578
Author(s):  
I Olivera-Valle ◽  
M C Latorre ◽  
M Calvo ◽  
B Gaspar ◽  
C Gómez-Oro ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Trichomonas Vaginalis ◽  
Large Scale ◽  
Ex Vivo ◽  
Detection System ◽  
Computer Assisted ◽  
Murine Models ◽  
Healthy Human ◽  
Sperm Analysis

Abstract STUDY QUESTION What is the vaginal polymorphonuclear (PMN) spermicidal mechanism to reduce the excess of sperm? SUMMARY ANSWER We show that PMNs are very efficient at killing sperm by a trogocytosis-dependent spermicidal activity independent of neutrophil extracellular traps (NETs). WHAT IS KNOWN ALREADY Trogocytosis has been described as an active membrane exchange between immune cells with a regulatory purpose. Recently, trogocytosis has been reported as a mechanism which PMNs use to kill tumour cells or Trichomonas vaginalis. STUDY DESIGN, SIZE, DURATION We used in vivo murine models and human ex vivo sperm and PMNs to investigate the early PMN–sperm response. PARTICIPANTS/MATERIALS, SETTING, METHODS We set up a live/dead sperm detection system in the presence of PMNs to investigate in vivo and ex vivo PMN-spermicidal activity by confocal microscopy, flow cytometry and computer-assisted sperm analysis (SCA). MAIN RESULTS AND THE ROLE OF CHANCE We revealed that PMNs are highly efficient at killing sperm by way of a NETs-independent, contact-dependent and serine proteases-dependent engulfment mechanism. PMNs ‘bite’ sperm and quickly reduce sperm motility (within 5 min) and viability (within 20 min) after contact. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION This study was conducted using murine models and healthy human blood PMNs; whether it is relevant to human vaginal PMNs or to cases of infertility is unknown. WIDER IMPLICATIONS OF THE FINDINGS Vaginal PMNs attack and immobilize excess sperm in the vagina by trogocytosis because sperm are exogenous and may carry pathogens. Furthermore, this mechanism of sperm regulation has low mucosal impact and avoids an exacerbated inflammatory response that could lead to mucosal damage or infertility. STUDY FUNDING/COMPETING INTEREST(S) This work was partially supported by Ministry of Economy and Competitiveness ISCIII-FIS grants, PI16/00050, and PI19/00078, co-financed by ERDF (FEDER) Funds from the European Commission, ‘A way of making Europe’ and IiSGM intramural grant II-PI-MRC-2017. M.R. holds a Miguel Servet II contract (CPII14/00009). M.C.L. holds IiSGM intramural contract. There are no competing interests.

IMMU-29. B-CELL-BASED VACCINE PRODUCES GLIOBLASTOMA-REACTIVE ANTIBODIES THAT CONTRIBUTE TO TUMOR CLEARANCE

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi98-vi98
Author(s):  
Brandyn Castro ◽  
Mark Dapash ◽  
David Hou ◽  
Aida Rashidi ◽  
Deepak Kanojia ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Humoral Immunity ◽  
Ex Vivo ◽  
Murine Models ◽  
Effector Functions ◽  
Tumor Bearing ◽  
Tumor Clearance ◽  
Cellular And Humoral Immunity

Abstract Glioblastomas (GBM) are characterized by a strong immunosuppressive environment, contributing to their poor prognosis and limited therapeutic response to immunotherapies. B-cells represent a unique opportunity to promote immunotherapy due to their potential to kill tumors by both cellular and humoral immunity. To generate our B-cell-based vaccine (BVax) platform, we activated 41BBL+ B cells from tumor bearing mice or GBM patient blood with BAFF, CD40, and IFNg. We have previously demonstrated that BVax potentiates radiation therapy, temozolomide and checkpoint blockade in murine models of GBM via enhancement of CD8+ T-cell based immunity. The aim of this current study is to evaluate the humoral effector functions of BVax. We examined the antibody (Ab) repertoire in vivo from serum of tumor-bearing B-cell knockout mice treated with BVax or by ex vivo stimulation of patient-derived BVax. Upon systemic administration, BVax infiltrates the tumor where it differentiates into plasmablasts. Murine BVax- and BNaive-derived serum immunoglobulin generated in vivo showed that the majority of murine BVax-derived Ab were IgG isotype, while BNaive mainly produced IgM isotype. Transfer of IgG from BVax treated mice directly into tumors of recipient animals significantly prolonged their survival, demonstrating anti-tumor cytotoxicity directly through humoral immunity. Patient-derived BVax activated ex vivo showed a plasmablast phenotype and the Ab repertoire supports the previous findings seen in our murine model. Our work suggests BVax-derived IgGs role in antibody-dependent cellular cytotoxicity and improved survival in murine models. This function, in addition to its role in cellular immunity against GBM, renders BVax a potentially effective alternative immunotherapeutic option for GBM patients.

scholarly journals Polarization sensitive optical coherence tomography with single input for imaging depth-resolved collagen organizations

2021 ◽  
Vol 10 (1) ◽  
Author(s):  
Peijun Tang ◽  
Mitchell A. Kirby ◽  
Nhan Le ◽  
Yuandong Li ◽  
Nicole Zeinstra ◽  
...  
Keyword(s):  
Optical Coherence Tomography ◽  
Structural Integrity ◽  
Ex Vivo ◽  
Polarization State ◽  
Transmission Model ◽  
Optical Coherence ◽  
Healthy Human ◽  
Collagen Organization ◽  
Single Input

AbstractCollagen organization plays an important role in maintaining structural integrity and determining tissue function. Polarization-sensitive optical coherence tomography (PSOCT) is a promising noninvasive three-dimensional imaging tool for mapping collagen organization in vivo. While PSOCT systems with multiple polarization inputs have demonstrated the ability to visualize depth-resolved collagen organization, systems, which use a single input polarization state have not yet demonstrated sufficient reconstruction quality. Herein we describe a PSOCT based polarization state transmission model that reveals the depth-dependent polarization state evolution of light backscattered within a birefringent sample. Based on this model, we propose a polarization state tracing method that relies on a discrete differential geometric analysis of the evolution of the polarization state in depth along the Poincare sphere for depth-resolved birefringent imaging using only one single input polarization state. We demonstrate the ability of this method to visualize depth-resolved myocardial architecture in both healthy and infarcted rodent hearts (ex vivo) and collagen structures responsible for skin tension lines at various anatomical locations on the face of a healthy human volunteer (in vivo).

Human erythroid cells produced ex vivo at large scale differentiate into red blood cells in vivo

2002 ◽  
Vol 20 (5) ◽  
pp. 467-472 ◽  
Author(s):  
Thi My Anh Neildez-Nguyen ◽  
Henri Wajcman ◽  
Michael C. Marden ◽  
Morad Bensidhoum ◽  
Vincent Moncollin ◽  
...  
Keyword(s):  
Red Blood Cells ◽  
Blood Cells ◽  
Large Scale ◽  
Ex Vivo ◽  
Erythroid Cells

scholarly journals Evaluation of persistence and fate of ex vivo edited-HSC modified with donor template and Its role in correcting Sickle Cell Disease

2021 ◽  
Author(s):  
Sowmya Pattabhi ◽  
Samantha N Lotti ◽  
Mason P Berger ◽  
David J Rawlings
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Ex Vivo ◽  
Cell Disease ◽  
Gene Correction ◽  
Healthy Human ◽  
Peripheral Blood Stem Cells ◽  
Donor Template

Sickle cell disease (SCD) is caused by a single nucleotide transversion in exon 1 of the HBB gene that changes the hydrophobicity of adult globin (βA), leading to substantial morbidity and reduced lifespan. Ex vivo autologous gene editing utilizing co-delivery of a designer nuclease along with a DNA donor template allows for precise homology-directed repair (HDR). These gene corrected cells when engrafted into the bone marrow (BM) can prove to be therapeutic and serves as an alternative to HLA-matched BM transplantation. In the current study, we extensively explored the role of single stranded oligonucleotide (ssODN) and recombinant adeno-associated 6 (rAAV6) donor template delivery to introduce a codon-optimized change (E6optE) or a sickle mutation (E6V) change following Crispr/Cas9-mediated cleavage of HBB in healthy human mobilized peripheral blood stem cells (mPBSCs). We achieved efficient HDR in vitro in edited cells and observed robust human CD45+ engraftment in the BM of NBSGW mice at 16-17 weeks. Notably, recipients of ssODN-modified HSC exhibited a significantly higher proportion of HDR-modified cells within individual BM, CD34+ and CD235+ compartments of both E6optE and E6V cohorts. We further assessed key functional outcomes including RNA transcripts analysis and globin sub-type expression. Our combined findings demonstrate the capacity to achieve clinically relevant HDR in vitro and in vivo using both donor template delivery method. The use of ssODN donor template-delivery is consistently associated with higher levels of gene correction in vivo as demonstrated by sustained engraftment of HDR-modified HSC and erythroid progeny. Finally, the HDR-based globin protein expression was significantly higher in the E6V ssODN-modified animals compared to the rAAV6-modified animals confirming that the ssODN donor template delivery outperforms rAAV6-donor template delivery.

scholarly journals Storage-Induced Micro-Erythrocytes Are Rapidly Cleared from Recipient Circulation and Predict Transfusion Recovery

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 717-717 ◽  
Author(s):  
Camille Roussel ◽  
Alexandre Morel ◽  
Michaël Dussiot ◽  
Mickael MARIN ◽  
Martin Colard ◽  
...  
Keyword(s):  
Research Funding ◽  
Ex Vivo ◽  
Retention Rate ◽  
Storage Duration ◽  
Healthy Human ◽  
Human Spleen ◽  
Inflammatory Monocytes ◽  
Human Volunteers ◽  
Reduced Surface

Background Hypothermic storage of red blood cell (RBC) concentrates for up to 42 days is associated with biochemical, molecular, morphological, and mechanical modifications. This "storage lesion" increases with storage duration and is associated with increased clearance of transfused storage-damaged RBCs from the recipient's circulation in the first few hours post-transfusion. This rapid clearance reduces transfusion efficacy, but how it occurs is not fully elucidated. RBCs with reduced surface area called "storage-induced micro-erythrocytes" (SMEs) were recently described. Their proportion increases from 2% to 23% during storage. Their reduced surface-to-volume ratio is expected to induce rapid mechanical clearance by the spleen. We aimed to evaluate whether SMEs can be used as a marker of transfusion efficacy, if this subpopulation of RBCs is preferentially cleared by the spleen after transfusion, and if so, by which mechanisms. Methods We evaluated the proportion of SMEs in stored RBC concentrates in vitro using ImageStream and correlated it to the 51Chromium-labeled 24h post-transfusion recovery (24hPTR) in vivo in 31 healthy human volunteers. We then investigated the fate of SMEs during 8 ex vivo perfusions of human spleens (16 RBC concentrates stored for 35-42 days). Finally, we developed a mouse transfusion model to assess the fate of SMEs in vivo and determine their main mechanisms of clearance. Results The proportion of SMEs in RBC concentrates at day 42 of storage correlated negatively with 24hPTR in healthy volunteers (r=-0.42, P<0.01). When perfused ex vivo into human spleens, 15% of stored RBCs (35-42 days of storage) were cleared during the first 40 min of perfusion in a 2-step process: 7% of circulating RBCs disappeared in the first 2 min (1-2 passages through the spleen) while 8% were cleared between 10 and 40 min after initiating perfusion (>5 passages through the spleen). The percentage of SMEs correlated with splenic retention rate ex vivo (r=0.46, p<0.05). Morphological analysis of 6 stored RBC concentrates showed a mean decrease in the proportion of SMEs from 20.2% to 7.8% between the beginning and end of splenic perfusions. In our mouse transfusion model, SMEs accumulated during RBC storage. The 24hPTR also decreased with storage duration (64% on Day 14 vs. 95% on Day 1). The decrease in 24hPTR of long-stored RBCs was mostly due to clearance of the SME subpopulation. SME and morphologically normal long-stored RBC subpopulations displayed clearances of 83% and 13%, respectively. Stored RBCs accumulated predominantly in the spleen post-transfusion, and were mainly ingested by macrophages. In macrophage-depleted mice, 24hPTR improved (from 64% to 79%), splenic accumulation and clearance of SMEs were delayed, and the proportion of inflammatory monocytes increased and mediated clearance. In splenectomized mice, clearance of SMEs was not delayed, but increased accumulation was observed in the liver and bone marrow, and increased erythrophagocytosis by inflammatory monocytes was also observed. Conclusions We show that the proportion of SMEs correlates with 24hPTR in healthy human volunteers and with retention in human spleens perfused ex vivo. In vivo mouse data confirms these findings, showing that SMEs are cleared from the recipient circulation during the 24h following transfusion. Clearance of SMEs is delayed in macrophage-depleted mice, suggesting a central role of macrophages in this process. The human spleen is also likely to clear SMEs from the recipient's circulation, as suggested by experiments with human spleens perfused ex vivo. However, the spleen is not required, because SME clearance is not affected in splenectomized mice. This suggests that other organs may compensate to remove SMEs and highlights the importance of eliminating these morphologically-altered RBCs. Finally, quantification of SMEs is an operator-independent, reproducible marker of transfusion efficacy. It can be used to assess the potential of new processes to prepare and store RBC concentrates. Pre-transfusion quantification of SMEs could benefit chronically transfused patients, for whom improved transfusion efficacy is expected to reduce transfusion-induced iron overload. Disclosures Roussel: Zimmer Biomet: Research Funding. MARIN:Zimmer Biomet: Research Funding. Spitalnik:Hemanext: Membership on an entity's Board of Directors or advisory committees; Tioma, Inc.: Consultancy. Hermine:AB science: Consultancy, Equity Ownership, Honoraria, Research Funding; Celgene: Research Funding; Novartis: Research Funding. Buffet:Zimmer Biomet: Research Funding. Amireault:Zimmer Biomet: Research Funding.

scholarly journals Capacitation-Induced Mitochondrial Activity Is Required for Sperm Fertilizing Ability in Mice by Modulating Hyperactivation

2021 ◽  
Vol 9 ◽  
Author(s):  
María Milagros Giaccagli ◽  
Matías Daniel Gómez-Elías ◽  
Jael Dafne Herzfeld ◽  
Clara Isabel Marín-Briggiler ◽  
Patricia Sara Cuasnicú ◽  
...  
Keyword(s):  
Mitochondrial Activity ◽  
Computer Assisted ◽  
Specific Staining ◽  
Sperm Analysis ◽  
Functional Changes ◽  
Fertilizing Ability ◽  
Sperm Fertilizing Ability

To become fully competent to fertilize an egg, mammalian sperm undergo a series of functional changes within the female tract, known as capacitation, that require an adequate supply and management of energy. However, the contribution of each ATP generating pathway to sustain the capacitation-associated changes remains unclear. Based on this, we investigated the role of mitochondrial activity in the acquisition of sperm fertilizing ability during capacitation in mice. For this purpose, the dynamics of the mitochondrial membrane potential (MMP) was studied by flow cytometry with the probe tetramethylrhodamine ethyl ester (TMRE). We observed a time-dependent increase in MMP only in capacitated sperm as well as a specific staining with the probe in the flagellar region where mitochondria are confined. The MMP rise was prevented when sperm were exposed to the mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazine (CCCP) or the protein kinase A (PKA) inhibitor H89 during capacitation, indicating that MMP increase is dependent on capacitation and H89-sensitive events. Results showed that whereas nearly all motile sperm were TMRE positive, immotile cells were mostly TMRE negative, supporting an association between high MMP and sperm motility. Furthermore, CCCP treatment during capacitation did not affect PKA substrate and tyrosine phosphorylations but produced a decrease in hyperactivation measured by computer assisted sperm analysis (CASA), similar to that observed after H89 exposure. In addition, CCCP inhibited the in vitro sperm fertilizing ability without affecting cumulus penetration and gamete fusion, indicating that the hyperactivation supported by mitochondrial function is needed mainly for zona pellucida penetration. Finally, complementary in vivo fertilization experiments further demonstrated the fundamental role of mitochondrial activity for sperm function. Altogether, our results show the physiological relevance of mitochondrial functionality for sperm fertilization competence.

TMOD-17. A NOVEL GLIOBLASTOMA INVASION MODEL USING HUMAN BRAIN SLICE CULTURES

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi219-vi219
Author(s):  
Vidyha Ravi ◽  
Kevin Joseph ◽  
Jürgen Beck ◽  
Oliver Schnell ◽  
Ulrich Hofmann ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Tumour Microenvironment ◽  
Human Model ◽  
Model Organisms ◽  
Murine Models ◽  
Loss Of Function ◽  
High Concentration ◽  
Ample Evidence ◽  
Human Microglia

Abstract OBJECTIVE Glioblastoma (GBM) is among the most common of malignant brain tumours, with a median post-surgical survival of less than one year. Over the past several decades, therapies that appeared promising in mice models have failed during clinical trials due to the differences encountered during translation of research from model organisms to humans. To partially mitigate these difficulties in translation, we present a human cortical organotypic culture based GBM model, which allows us to manipulate individual components of the tumour environment in order to investigate the influence of different cell types in the immunosuppressive tumour microenvironment. METHODS Human neocortical tissue (at least 2 cm away from the tumour core) or entry cortex from epilepsy surgery guided by intraoperative neuro navigation, was cultured for up to 14 days post resection using an optimized medium. The cultured tissue was further injected with patient derived human GBM cells to create an ex vivo human model of glioblastoma model. The role of astrocytes in the tumour microenvironment was studied using microglia loss of function model. RESULTS Our established human neo-cortical slice model can recapitulate an in-vivo characteristics of glioblastoma from functional and imaging aspect. Our data corroborate differences between astrocytes in human and murine models in different reactive states, shows that the glioblastoma microenvironment can be difficult to be accurately modelled using murine models. Results from our human microglia depletion model, provided ample evidence that complex interaction of astrocytes and microglia cells, promotes an immunosuppressive environment in Glioblastoma by releasing high concentration of IL10 and TGFbeta (p< 0.001). CONCLUSION Our model therefore has potential applications to the fields of neuroscience, neuro-oncology, and pharmacotherapy.

Endogenous biosynthesis of thromboxane and prostacyclin in 2 distinct murine models of atherosclerosis

Blood ◽  
2000 ◽  
Vol 96 (12) ◽  
pp. 3823-3826 ◽  
Author(s):  
Domenico Praticò ◽  
Tillmann Cyrus ◽  
Hongwei Li ◽  
Garret A. FitzGerald
Keyword(s):  
Ex Vivo ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Thromboxane B2 ◽  
Murine Models ◽  
En Face ◽  
Density Lipoprotein Receptor ◽  
Potent Vasoconstrictor ◽  
Deficient Mice

Abstract Thromboxane A2 is a potent vasoconstrictor and platelet agonist; prostacyclin is a potent platelet inhibitor and vasodilator. Altered biosynthesis of these eicosanoids is a feature of human hypercholesterolemia and atherosclerosis. This study examined whether in 2 murine models of atherosclerosis their levels are increased and correlated with the evolution of the disease. Urinary 2,3-dinor thromboxane B2 and 2,3-dinor-6-keto prostaglandin F1α, metabolites of thromboxane and prostacyclin, respectively, were assayed in apoliprotein E (apoE)-deficient mice on chow and low-density lipoprotein receptor (LDLR)-deficient mice on chow and a Western-type diet. Atherosclerosis lesion area was measured by en face method. Both eicosanoids increased in apoE-deficient mice on chow and in LDLR-deficient mice on a high-fat diet, but not in LDLR-deficient mice on chow by the end of the study. Aspirin suppressed ex vivo platelet aggregation, serum thromboxane B2, and 2,3-dinor thromboxane B2, and significantly reduced the excretion of 2,3-dinor-6-keto prostaglandin F1α in these animals. This study demonstrates that thromboxane as well as prostacyclin biosynthesis is increased in 2 murine models of atherogenesis and is secondary to increased in vivo platelet activation. Assessment of their generation in these models may afford the basis for future studies on the functional role of these eicosanoids in the evolution and progression of atherosclerosis.

Evaluation of colonisation resistance in stool of human donors using ex vivo, in vitro and in vivo assays

2017 ◽  
Vol 8 (2) ◽  
pp. 217-230 ◽  
Author(s):  
M.F. Galvão ◽  
R.W. Bastos ◽  
L.B. Acurcio ◽  
B.B. Nascimento ◽  
S.H.C. Sandes ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Faecal Microbiota ◽  
Healthy Human ◽  
In Vivo Assays ◽  
Enteropathogenic Bacteria ◽  
Histological Data ◽  
Lactobacillus Ruminis ◽  
Colonisation Resistance

The indigenous microbiota is the population of microorganisms normally present on the surface and mucosa of an individual, where it performs essential health functions, including the colonisation resistance (CR) against pathogens. To identify the bacteria responsible and the mechanisms involved in the CR, the germ-free (GF) animal model has been used, because in vitro studies cannot always be extrapolated to what occurs in vivo. In this study, ex vivo antagonism assays against seven enteropathogenic bacteria using stools from 15 healthy human donors confirmed that the CR showed individual variation. Using in vitro antagonism assays, 14 strains isolated from dominant faecal microbiota of donors with elevated CR were selected for mono-association in GF mice to test the in vivo antagonism against Salmonella enterica ser. Typhimurium. Mice mono-associated with Enterococcus hirae strain 8.2, Bacteroides thetaiotaomicron strain 16.2 and Lactobacillus ruminis strain 18.1 had significant reductions in faecal counts of the pathogen during the challenge. After five days of infection, the group associated with E. hirae 8.2 showed a reduction in the translocation of S. Typhimurium to the spleen, while the group associated with L. ruminis 18.1 presented an increased translocation to the liver. The histological data confirmed these results and revealed that the mice associated with E. hirae 8.2 showed fewer lesions on ileum and liver, compared to the damage caused by S. Typhimurium alone, while in mice associated with L. ruminis 18.1 there was significantly worse lesions. Concluding, from the dominant faecal microbiota from healthy human with high CR, through ex vivo, in vitro and in vivo assays, a bacterium was characterised for its high CR potential, being a candidate for probiotic use.

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