The impact of the herbicide glyphosate and its metabolites AMPA and MPA on the metabolism and functions of human blood neutrophils and their sex-dependent effects on reactive oxygen species and CXCL8/IL-8 production.

Significant levels of glyphosate, the world's most widely used herbicide, and its primary metabolites, AMPA and MPA, are detected in various human organs and body fluids, including blood. Several studies have associated the presence of glyphosate in humans with health problems, and effects on immune cells and their functions have been reported. However, the impact of this molecule and its metabolites on neutrophils, the most abundant leukocytes in the human bloodstream, is still poorly documented. We isolated neutrophils from human donor blood and investigated the effects of exposure to glyphosate, AMPA, and MPA on viability, energy metabolism, and essential antimicrobial functions in vitro. We observed that neutrophil viability was unaffected at the blood-relevant average concentrations of the general population and exposed workers, as well as at higher intoxication concentrations. Neutrophil energy metabolism was also not altered following exposure to the chemicals. However, while phagocytosis was unaffected, reactive oxygen species generation and CXCL8/IL-8 production were altered by exposure to the molecules. Alterations in function following exposure to glyphosate and metabolites differed according to the sex of the donors, which could be linked to glyphosate's known role as an endocrine disruptor. While ROS generation was increased in both sexes, male neutrophils exposed to glyphosate had increased intracellular production of CXCL8/IL-8, with no effect on female neutrophils. Conversely, exposure to the metabolites AMPA and MPA decreased extracellular production of this chemokine only in female neutrophils, with MPA also increasing intracellular production in male cells exposed to the chemoattractant N-formyl-methionine-leucyl-phenylalanine. Our study highlights the effects of glyphosate and its metabolites on the antimicrobial functions of neutrophils, which could be associated with health problems as future studies provide a better understanding of the risks associated with glyphosate use. Advances in knowledge will enable better and potentially stricter regulations to protect the public.

The follicle-stimulating hormone triggers rapid changes in mitochondrial structure and function in porcine cumulus cells.

Oocyte maturation is a key process during which the female germ cell undergoes resumption of meiosis and completes its preparation for embryonic development including cytoplasmic and epigenetic maturation. The cumulus cells directly surrounding the oocyte are involved in this process by transferring essential metabolites, such as pyruvate, to the oocyte. This process is controlled by cyclic adenosine monophosphate (cAMP)-dependent mechanisms recruited downstream of follicle-stimulating hormone (FSH) signaling in cumulus cells. As mitochondria have a critical but poorly understood contribution to this process, we defined the effects of FSH and high cAMP concentrations on mitochondrial dynamics and function in porcine cumulus cells. During in vitro maturation (IVM) of cumulus-oocyte complexes (COCs), we observed an FSH-dependent mitochondrial elongation shortly after stimulation that led to mitochondrial fragmentation 24 h later. Importantly, mitochondrial elongation was accompanied by decreased mitochondrial activity and a switch to glycolysis. During a pre-IVM culture step increasing intracellular cAMP, mitochondrial fragmentation was prevented. Altogether, the results demonstrate that FSH triggers rapid changes in mitochondrial structure and function in COCs involving cAMP.

Estrogens and endocrine-disrupting chemicals differentially impact the bioenergetic fluxes of mammary epithelial cells in two- and three-dimensional models.

Due to its sensitivity to hormonal signaling, the mammary gland is often referred to as a sentinel organ for the study of endocrine-disrupting chemicals (EDCs), environmental pollutants that can interfere with the estrogen signaling pathway and induce mammary developmental defects. If and how EDCs impact mammary epithelial cell metabolism has not yet been documented. Herein, to study how estrogens and EDCs modulate mammary gland metabolism, we performed bioenergetic flux analyses using mouse mammary epithelial organoids compared to cells grown in monolayer culture. Several EDCs were tested, including bisphenol A (BPA), its close derivative BPS, a new BPA replacement copolyester called TritanTM, and the herbicide glyphosate. We report that estrogens reprogrammed mammary epithelial cell metabolism differently when grown in two- and three-dimensional models. Specific EDCs were also demonstrated to alter bioenergetic fluxes, thus identifying a new potential adverse effect of these molecules. Notably, organoids were more sensitive to low EDC concentrations, highlighting them as a key model for screening the impact of various environmental pollutants. Mechanistically, transcriptomic analyses revealed that EDCs interfered with the regulation of estrogen target genes and the expression of metabolic genes in organoids. Furthermore, co-treatment with the anti-estrogen fulvestrant blocked these metabolic impacts of EDCs, suggesting that, at least partially, they act through modulation of the estrogen receptor activity. Finally, we demonstrate that mammary organoids can be used for long-term studies on EDC exposure to study alterations in organogenesis/morphogenesis and that past pregnancies can modulate the sensitivity of mammary epithelial organoids to specific EDCs. Overall, this study demonstrates that estrogens and EDCs modulate mammary epithelial cell metabolism in monolayer and organoid cultures. A better understanding of the metabolic impacts of EDCs will allow a better appreciation of their adverse effects on mammary gland development and function.

Platelet extracellular vesicles and their mitochondrial content improve the mitochondrial bioenergetics of cellular immune recipients.

BACKGROUND: Mitochondria play a critical role in the production of cell energy and the regulation of cell death. Therefore, mitochondria orchestrate numerous cell effector functions, including fine-tuning the immune system. While mitochondria are mainly found intracellularly, they can escape the confine of the cell during the process of extracellular vesicle release. Platelets patrol blood vessels to ensure vasculature integrity and to support the immune system. In blood, platelets are the primary source of circulating mitochondria. Activated platelets produce extracellular vesicles, including a subset of mitochondria-containing vesicles. STUDY DESIGN AND METHODS: We characterized mitochondrial functions in platelet-derived extracellular vesicles, and examined whether they could impact the bioenergetics of cellular immune recipients using an extracellular flux analyzer to measure real-time bioenergetics. RESULTS: We validated that extracellular vesicles derived from activated platelets contain the necessary mitochondrial machinery to respirate and generate energy. Moreover, neutrophils and monocytes efficiently captured platelet-derived extracellular vesicles, enhancing their mitochondrial fitness. This process required functional mitochondria from donor platelets, as it was abolished by the inactivation of extracellular mitochondria using mitochondrial poison. DISCUSSION: Together, the data suggest that extracellular mitochondria produced by platelets may support other metabolic functions through transcellular bioenergetics.

Can intraoperative radiation dose in percutaneous posterior thoracolumbar internal fixation be reduced by impedancemetry-guided pedicle sighting? A prospective randomized study.

INTRODUCTION: Percutaneous spine surgery is on the rise; the main drawback is iterative irradiation of the care team in theater. The aim of the present study was to compare intraoperative radiation dose in percutaneous posterior thoracolumbar internal fixation (PPTLIF) using impedancemetry-guided pedicle sighting by the PediGuard device (SpineGuard®) versus gold-standard free-hand sighting. MATERIAL AND METHODS: A single-center, single-surgeon continuous prospective randomized study was conducted from September 2017 to April 2018. Dose-area product (DAP, in cGy.cm2) was recorded at the end of pedicle sighting and end of surgery in the free-hand control group and the impedancemetry group. Pedicle screw position was studied on postoperative CT scan. RESULTS: Sixteen patients were included in either group after 2 had been excluded. The groups were comparable for age, gender, body-mass index (BMI), indication and number of instrumented levels. Mean DPA at end of sighting and end of procedure was respectively 147.4 cGy.cm2 and 230.9 cGy.cm2 in the control group and 171.1 cGy.cm2 and 280.7 cGy.cm2 in the PediGuard group (p> 0.05). Screw positioning on CT was comparable in the 2 groups. CONCLUSION: In the present study, the PediGuard device did not reduce intraoperative radiation dose. The correlation between radiation dose and BMI was confirmed. LEVEL OF EVIDENCE: II; prospective randomized study.

The Balance between p53 Isoforms Modulates the Efficiency of HIV-1 Infection in Macrophages.

Several host factors influence HIV-1 infection and replication. The p53-mediated antiviral role in monocyte-derived macrophages (MDMs) was previously highlighted. Indeed, an increase in p53 level results in a stronger restriction against HIV-1 early replication steps through SAMHD1 activity. In this study, we investigated the potential role of some p53 isoforms in HIV-1 infection. Transfection of isoform-specific small interfering RNA (siRNA) induced distinctive effects on the virus life cycle. For example, in contrast to an siRNA targeting all isoforms, a knockdown of Δ133p53 transcripts reduced virus replication in MDMs that was correlated with a decrease in phosphorylated inactive SAMHD1. Combination of Δ133p53 knockdown and nutlin-3, a pharmacological inhibitor of MDM2 that stabilizes p53, further reduced susceptibility of MDMs to HIV-1 infection, thus suggesting an inhibitory role of Δ133p53 toward p53 antiviral activity. In contrast, p53ß knockdown in MDMs increased the viral production independently of SAMHD1. Moreover, experiments with a Nef-deficient virus showed that this viral protein plays a protective role against the antiviral environment mediated by p53. Finally, HIV-1 infection affected the expression pattern of p53 isoforms by increasing p53ß and p53γ mRNA levels while stabilizing the protein level of p53α and some isoforms from the p53ß subclass. The balance between the various p53 isoforms is therefore an important factor in the overall susceptibility of macrophages to HIV-1 infection, fine-tuning the p53 response against HIV-1. This study brings a new understanding of the complex role of p53 in virus replication processes in myeloid cells. IMPORTANCE As of today, HIV-1 infection is still considered a global pandemic without a functional cure, partly because of the presence of stable viral reservoirs. Macrophages constitute one of these cell reservoirs, contributing to the viral persistence. Studies investigating the host factors involved in cell susceptibility to HIV-1 infection might lead to a better understanding of reservoir formation and will eventually allow the development of an efficient cure. Our team previously showed the antiviral role of p53 in macrophages, which acts by compromising the early steps of HIV-1 replication. In this study, we demonstrate the involvement of p53 isoforms, which regulate p53 activity and define the cellular environment influencing viral replication. In addition, the results concerning the potential role of p53 in antiviral innate immunity could be transposed to other fields of virology and suggest that knowledge in oncology can be applied to HIV-1 research.

Morbidity and clinicoradiological outcomes of anterior lumbar arthrodesis using tantalum intervertebral implants.

PURPOSE: The objective of this study was to assess the morbidity of Anterior Lumbar Interbody Fusion (ALIF) using an intervertebral tantalum implant. Tantulum is an extremely porous metallic material which possesses properties of osseointegration, osteoinduction and osteoconduction while offering superior primary stability, compared to other materials more commonly used (polyether ether ketone or PEEK, titanium). Perioperative morbidity, short-term functional outcomes (2 years) and radiographic impaction of implants were also analysed. METHODS: This retrospective monocentric study involved 94 patients operated on between 2014 and 2017 for degenerative disc disease (75%), degenerative spondylolisthesis (3%) or isthmic lytic spondylolisthesis (22%). Sixty-five patients (69%) had isolated ("stand-alone") ALIF procedures, 24 (26%) with associated anterior osteosynthesis and 5 (5%) with associated posterior osteosynthesis. A Kaplan-Meier survival curve was established with surgical revision listed as the main criterion for failure. Perioperative complications were identified. The clinical evaluation at the last follow-up used a Visual Analogue Scale for radicular pain (VAS-R), for lumbar pain (VAS-L) and the Oswestry Disability Index (ODI) score. The impactions, assessed on x-rays, were divided into 2 groups according to severity in order to establish risk factors (RF). RESULTS: The primary objective showed a 2-year survival rate of 94% (95% CI [0.88; 0.99]). Two patients had early surgical revision for impaction and 4 patients had late surgical revision for pseudarthrosis. The rate of perioperative complications was 8.5%, mostly due to vascular causes. At the average follow-up of 33 months (24-59), the clinical results were significantly improved and the impaction rate was 36% in the immediate postoperative period (IPO) and 47% at one year. CONCLUSION: ALIF using an intervertebral tantalum implant is a reliable, reproducible and low morbidity technique. However, it is accompanied by a significant rate of immediate and secondary impaction but without any resounding influence on short-term clinical outcomes. LEVEL OF EVIDENCE: IV.

Thymidylate synthase is essential for efficient HIV-1 replication in macrophages.

Thymidylate synthase (TS) is a key enzyme in nucleotide biosynthesis. A study performed by our group on human monocyte-derived macrophages (MDMs) infected with HIV-1 showed that many enzymes related to the folate cycle pathway, such as TS, are upregulated in productively infected cells. Here, we suggest that TS is essential for an effective HIV-1 infection in MDMs. Indeed, a TS specific small interfering RNA (siRNA) as well as the TS specific inhibitor Raltitrexed (RTX) caused a reduction in productively infected cells. Quantitative PCR analysis showed that this treatment decreased the efficacy of the early steps of the viral cycle. The RTX inhibitory effect was counteracted by dNTP addition. These results suggest that TS is essential for the early stages of HIV-1 infection by providing optimal dNTP concentrations in MDMs. TS and its related pathway may thus be considered as a potential therapeutic target for HIV-1 treatment.

Tantalum implants for posterior lumbar interbody fusion: A safe method at medium-term follow-up?

INTRODUCTION: Intervertebral implants increase stability and improve results in lumbar interbody fusion (LIF). The aim of the present study was to assess clinical and radiological results of posterior lumbar interbody fusion (PLIF) using a tantalum intervertebral implant without associated interbody bone graft. MATERIEL AND METHODS: A single-center retrospective study included 52 cases of single-level PLIF, using 2 tantalum intervertebral cages, without interbody bone graft: 42 for degenerative disc disease, 10 for isthmic spondylolisthesis. Minimum follow-up was 2 years. Clinical assessment used a visual analog (pain) scale (VAS), the Oswestry Disability Index (ODI) and the Roland Morris (RM) scale. Tantalum osseointegration and intersegment mobility were assessed on static and dynamic X-ray. RESULTS: Forty-nine patients were included, with a mean 55months' follow-up (range, 25-74months). VAS, ODI and RM scores showed significant improvement at last-follow-up, at 4, 30 and 28 points respectively. There was no mechanical failure on static X-ray; all patients had less than 5° mobility on dynamic X-ray at last follow-up. DISCUSSION: PLIF with tantalum intervertebral implant without interbody bone graft provided satisfactory clinical and radiological results at medium-term follow-up. The present findings showed reliable primary stability and osseointegration of the tantalum implant. LEVEL OF EVIDENCE: IV.

Host mRNA decay proteins influence HIV-1 replication and viral gene expression in primary monocyte-derived macrophages.

BACKGROUND: Mammalian cells harbour RNA quality control and degradative machineries such as nonsense-mediated mRNA decay that target cellular mRNAs for clearance from the cell to avoid aberrant gene expression. The role of the host mRNA decay pathways in macrophages in the context of human immunodeficiency virus type 1 (HIV-1) infection is yet to be elucidated. Macrophages are directly infected by HIV-1, mediate the dissemination of the virus and contribute to the chronic activation of the inflammatory response observed in infected individuals. Therefore, we characterized the effects of four host mRNA decay proteins, i.e., UPF1, UPF2, SMG6 and Staufen1, on viral replication in HIV-1-infected primary monocyte-derived macrophages (MDMs). RESULTS: Steady-state expression levels of these host mRNA decay proteins were significantly downregulated in HIV-1-infected MDMs. Moreover, UPF2 and SMG6 inhibited HIV-1 gene expression in macrophages to a similar level achieved by SAMHD1, by directly influencing viral genomic RNA levels. Staufen1, a host protein also involved in UPF1-dependent mRNA decay and that acts at several HIV-1 replication steps, enhanced HIV-1 gene expression in MDMs. CONCLUSIONS: These results provide new evidence for roles of host mRNA decay proteins in regulating HIV-1 replication in infected macrophages and can serve as potential targets for broad-spectrum antiviral therapeutics.

Expression of MDM2 in Macrophages Promotes the Early Postentry Steps of HIV-1 Infection through Inhibition of p53.

The molecular basis for HIV-1 susceptibility in primary human monocyte-derived macrophages (MDMs) was previously evaluated by comparing the transcriptome of infected and bystander populations. Careful analysis of the data suggested that the ubiquitin ligase MDM2 acted as a positive regulator of HIV-1 replication in MDMs. In this study, MDM2 silencing through transcript-specific small interfering RNAs in MDMs induced a reduction in HIV-1 reverse transcription and integration along with an increase in the expression of p53-induced genes, including CDKN1A Experiments with Nutlin-3, a pharmacological inhibitor of MDM2 p53-binding activity, showed a similar effect on HIV-1 infection, suggesting that the observed restriction in HIV-1 production results from the release/activation of p53 and not the absence of MDM2 per se Knockdown and inhibition of MDM2 also both correlate with a decrease in the Thr592-phosphorylated inactive form of SAMHD1. The expression level of MDM2 and the p53 activation status are therefore important factors in the overall susceptibility of macrophages to HIV-1 infection, bringing a new understanding of signaling events controlling the process of virus replication in this cell type.IMPORTANCE Macrophages, with their long life span in vivo and their resistance to HIV-1-mediated cytopathic effect, might serve as viral reservoirs, contributing to virus persistence in an infected individual. Identification of host factors that increase the overall susceptibility of macrophages to HIV-1 might provide new therapeutic targets for the efficient control of viral replication in these cells and limit the formation of reservoirs in exposed individuals. In this study, we demonstrate the importance of p53 regulation by MDM2, which creates a cellular environment more favorable to the early steps of HIV-1 replication. Moreover, we show that p53 stabilization reduces virus infection in human macrophages, highlighting the important role of p53 in antiviral immunity.

Global Mapping of the Macrophage-HIV-1 Transcriptome Reveals that Productive Infection Induces Remodeling of Host Cell DNA and Chromatin.

It has been proposed that macrophages could serve as long-lived compartments for HIV-1 infection under in vivo situations because these cells are resistant to the virus-mediated cytopathic effect, produce progeny virus over extended periods of time and are localized in tissues that are often less accessible by treatment. Comprehensive experimental studies are thus needed to characterize the HIV-1-induced modulation of host genes in these myeloid lineage cells. To shed light on this important issue, we performed comparative analyses of mRNA expression levels of host genes in uninfected bystander and HIV-1-infected human macrophages using an infectious reporter virus construct coupled with a large-scale RNA sequencing approach. We observed a rapid differential expression of several host factors in the productively infected macrophage population including genes regulating DNA replication factors and chromatin remodeling. A siRNA-mediated screening study to functionally identify host determinants involved in HIV-1 biology has provided new information on the virus molecular regulation in macrophages.

HIV-1-Mediated BAFF Secretion in Macrophages Does Not Require Endosomal TLRs, Type-I IFN, and Nef, but Depends on the Cellular Phenotype Status.

HIV-1 infection is characterized by persistent viral replication, chronic immune activation, and CD4(+) T cell depletion. Moreover, several immune dysfunctions are observed in cells that are not targeted by the virus, such as B cells. Some B cell abnormalities include hypergammaglobulinemia, nonspecific B cell activation, class switching, increased cell turnover, breakage of tolerance, and a loss of the capacity to generate and maintain memory. Several cytokines and growth factors that are increased in the serum of HIV-1-infected individuals have been suggested to directly or indirectly trigger B cell activation, and one of these is BAFF. In this study, we investigate the ability of fully competent (R5-tropic) HIV-1 to induce BAFF production by monocyte-derived macrophages (MDMs). We demonstrate here that HIV-1 drives BAFF production in MDMs in a type-I IFN- and TLR-independent manner. Moreover, we determine that HIV-1 Nef accessory protein is dispensable in BAFF upregulation as a nef-deleted HIV-1 strain is still able to increase BAFF at levels similar to the wild type strain. Finally, we show that the macrophage phenotype status affects HIV-1 replication and BAFF induction, as both were abrogated in MDMs displaying a M1 phenotype. This study provides new useful information about the increased levels of BAFF observed during HIV-1 infection and highlights the importance of macrophages as a source of BAFF, a phenomenon that might contribute to B cell dysfunctions at inflammatory tissue sites in infected individuals.