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International audience; Ice losses from the Greenland and Antarctic ice sheets have accelerated since the 1990s, accounting for a significant increase in the global mean sea level. Here, we present a new 29-year record of ice sheet mass balance from 1992 to 2020 from the Ice Sheet Mass Balance Inter-comparison Exercise (IMBIE). We compare and combine 50 independent estimates of ice sheet mass balance derived from satellite observations of temporal changes in ice sheet flow, in ice sheet volume, and in Earth's gravity field. Between 1992 and 2020, the ice sheets contributed 21.0±1.9 mm to global mean sea level, with the rate of mass loss rising from 105 Gt yr−1 between 1992 and 1996 to 372 Gt yr−1 between 2016 and 2020. In Greenland, the rate of mass loss is 169±9 Gt yr−1 between 1992 and 2020, but there are large inter-annual variations in mass balance, with mass loss ranging from 86 Gt yr−1 in 2017 to 444 Gt yr−1 in 2019 due to large variability in surface mass balance. In Antarctica, ice losses continue to be dominated by mass loss from West Antarctica (82±9 Gt yr−1) and, to a lesser extent, from the Antarctic Peninsula (13±5 Gt yr−1). East Antarctica remains close to a state of balance, with a small gain of 3±15 Gt yr−1, but is the most uncertain component of Antarctica's mass balance. The dataset is publicly available at https://doi.org/10.5285/77B64C55-7166-4A06-9DEF-2E400398E452 (IMBIE Team, 2021).

Understanding the behavior and ecology of local malaria vectors is essential for the effectiveness of the commonly used vector-targeted malaria control tools in areas of low malaria transmission. This study was conducted to determine species composition, biting behavior and infectivity of the major Anopheles vectors of Plasmodium falciparum in low transmission settings in central Senegal. Adult mosquitoes were collected using human landing catches during 2 consecutive nights and Pyrethrum Spray Catches in 30–40 randomly selected rooms, from July 2017 to December 2018 in 3 villages. Anopheline mosquitoes were morphologically identified using conventional keys; their reproductive status assessed by ovary dissections, and a sub-sample of Anopheles gambiae s.l. were identified to species level using polymerase chain reaction (PCR). Plasmodium sporozoite infections were detected using real-time quantitative PCR. During this study 3684 Anopheles were collected of which 97% were An. gambiae s.l., 0.6% were Anopheles funestus, and 2.4% were Anopheles pharoensis. Molecular identification of 1,877 An. gambiae s.l. revealed a predominance of Anopheles arabiensis (68.7%), followed by Anopheles melas (28.8%), and Anopheles coluzzii (2.1%). The overall human-biting rate of An. gambiae s.l. was highest in the inland site of Keur Martin with 4.92 bites per person per night, while it was similar in the deltaic site, Diofior (0.51) and the coastal site, Mbine Coly (0.67). Parity rates were similar in An. arabiensis (45%) and An. melas (42%). Sporozoite infections were detected in both An. arabiensis and An. melas with the respective infection rates of 1.39% (N = 8) and 0.41% (N = 1). Results suggest that low residual malaria in central Senegal is transmitted by An. arabiensis and An. melas. Consequently, both vectors will need to be targeted as part of malaria elimination efforts in this area of Senegal.

Bacterial and fungal pathogens face various microenvironmental conditions during infection. In addition to acidosis, nutrient consumption, and hypercapnia, pathogen infections are associated with hypoxia, which is induced by bacterial and fungal respiration during the formation of foci of infection or biofilms. Consequently, the in vivo interaction between host immune cells and pathogens is anticipated to occur mainly under low-oxygen conditions. Various infectious disease models have reported that pathogens benefit from hypoxia, which dampens the oxygen-dependent antimicrobial activities of macrophages and neutrophils, such as the production of reactive oxygen species (ROS). Due to their dual respiration capacity (aerobic and anaerobic) or phenotypical adaptation (e.g., dormancy), pathogens have the capacity to survive and disseminate in the absence of oxygen. In addition, hypoxia modulates various mechanisms of pathogen virulence, promoting the dissemination of pathogens. Further investigations are still required to evaluate the relative importance of oxygen on the capacity of pathogens to invade and colonize host organs and to better understand alternative strategies developed by immune cells to circumvent pathogen dissemination in the absence of oxygen. Addressing this important and fundamental question in various models of infection may direct the development of innovative therapeutic strategies.

Summary paragraphNeutrophil degranulation plays a central role in their ability to kill pathogens but also to stimulate other immune cells1–3. Here we show that neutrophil degranulation, induced in hypoxia or upon Shigella infection in vitro and in vivo, leads to the release of polymers called neutrophil Proteoglycofili (PGF). PGF are mainly composed of granular proteins (myeloperoxidase, elastase, lactoferrin, cathelicidin, albumin) pre-stored in various types of granules, and chondroitin sulfate. PGF individual fibers have a diameter of 43.9 ± 20.3 nm and. They secreted by viable neutrophils and do not contain DNA, as opposed to NETs which contains also granular proteins, chondroitin sulfate in addition to chromatin, released upon neutrophil disintegration and cell death.We demonstrated that PGF block the growth of Shigella and other bacteria and degrade Shigella virulence factors. The degradation of the chondroitin sulfate polymers with testes hyaluronidases destabilizes PGF ultrastructure and abolishes its antimicrobial activity. Our results provide novel insights in the neutrophil degranulation process and open new doors for the investigation of PGF contribution to cytokines concentration gradient formation and adaptive immune cells activation. Further investigations are required to better appreciate the importance of this “sterile blaster” in infectious or inflammatory diseases.

A fluorogenic benzochalcone specifically labels live neutrophil granules in whole wild-type, GFP- or RFP-expressing zebrafish embryos and larvae., Neutrophil granules (NGs) are key components of the innate immune response and mark the development of neutrophilic granulocytes in mammals. However, there has been no specific fluorescent vital stain up to now to monitor their dynamics within a whole live organism. We rationally designed a benzochalcone fluorescent probe (HAB) featuring high tissue permeability and optimal photophysics such as elevated quantum yield, pronounced solvatochromism and target-induced fluorogenesis. Phenotypic screening identified HAB as the first cell- and organelle-specific small-molecule fluorescent tracer of NGs in live zebrafish larvae, with no labeling of other cell types or organelles. HAB staining was independent of the state of neutrophil activation, labeling NGs of both resting and phagocytically active neutrophils with equal specificity. By high-resolution live imaging, we documented the dynamics of HAB-stained NGs during phagocytosis. Upon zymosan injection, labeled NGs were rapidly recruited to the forming phagosomes. Despite being a reversible ligand, HAB could not be displaced by high concentrations of pharmacologically relevant competing chalcones, indicating that this specific labeling was the result of the HAB's precise physicochemical signature rather than a general feature of chalcones. However, one of the competitors was discovered as a promising interstitial fluorescent tracer illuminating zebrafish histology, similarly to BODIPY-ceramide. As a yellow-emitting histopermeable vital stain, HAB functionally and spectrally complements most genetically incorporated fluorescent tags commonly used in live zebrafish biology, holding promise for the study of neutrophil-dependent responses relevant to human physiopathology such as developmental defects, inflammation and infection. Furthermore, HAB intensely labeled isolated live human neutrophils at the level of granulated subcellular structures consistent with human NGs, suggesting that the labeling of NGs by HAB is not restricted to the zebrafish model but also relevant to mammalian systems.

Bacteria, including those that are pathogenic, have been generally classified according to their ability to survive and grow in the presence or absence of oxygen: aerobic and anaerobic bacteria, respectively. Strict aerobes require oxygen to grow (e.g., Neisseria), and strict anaerobes grow exclusively without, and do not survive oxygen exposure (e.g., Clostridia); aerotolerant bacteria (e.g., Lactobacilli) are insensitive to oxygen exposure. Facultative anaerobes (e.g., E. coli) have the unique ability to grow in the presence or in the absence of oxygen and are thus well-adapted to these changing conditions, which may constitute an underestimated selective advantage for infection. In the WHO antibiotic-resistant 'priority pathogens' list, facultative anaerobes are overrepresented (8 among 12 listed pathogens), consistent with clinical studies performed in populations particularly susceptible to infectious diseases. Bacteria aerobic respiratory chain plays a central role in oxygen consumption, leading to the formation of hypoxic infectious sites (infectious hypoxia). Facultative anaerobes have developed a wide diversity of aerotolerance and anaerotolerance strategies in vivo. However, at a single cell level, the modulation of the intracellular oxygen level in host infected cells remains elusive and will be discussed in this review. In conclusion, the ability of facultative bacteria to evolve in the presence or the absence of oxygen is essential for their virulence strategy and constitute a selective advantage. TAKE AWAY: Most life-threatening pathogenic bacteria are facultative anaerobes. Only facultative anaerobes are aerotolerant, anaerotolerant and capable of consuming O2 . Facultative anaerobes induce and are well adapted to cellular hypoxia.

Under closed-list proportional representation, a party's electoral list determines the order in which legislative seats are allocated to candidates. When candidates differ in their ability, parties face a trade-off between competence and incentives. Ranking candidates in decreasing order of competence ensures that elected politicians are most competent. Yet, party lists create incentives for candidates that may push parties not to rank candidates in decreasing competence order. We examine this trade-off in a game-theoretical model in which parties rank their candidate on a list, candidates choose their campaign effort, and the election is a team contest for multiple prizes. We show that the trade-off between competence and incentives depends on candidates' objective and the electoral environment. In particular, parties rank candidates in decreasing order of competence if candidates value enough post-electoral high offices or media coverage focuses on candidates at the top of the list.

Neutrophils are the most abundant circulating white blood cells and are the central players of the innate immune response. During their lifecycle, neutrophils mainly evolve under low oxygen conditions (0.1–4% O2), to which they are well adapted. Neutrophils are atypical cells since they are highly glycolytic and susceptible to oxygen exposure, which induces their activation and death through mechanisms that remain currently elusive. Nevertheless, nearly all studies conducted on neutrophils are carried out under atmospheric oxygen (21%), corresponding to hyperoxia. Here, we investigated the impact of hyperoxia during neutrophil purification and culture on neutrophil viability, activation and cytosolic protein content. We demonstrate that neutrophil hyper‐activation (CD62L shedding) is induced during culture under hyperoxic conditions (24 h), compared with neutrophils cultured under anoxic conditions. Spontaneous neutrophil extracellular trap (NET) formation is observed when neutrophils face hyperoxia during purification or culture. In addition, we show that maintaining neutrophils in autologous plasma is the preferred strategy to maintain their basal state. Our results show that manipulating neutrophils under hyperoxic conditions leads to the loss of 57 cytosolic proteins during purification, while it does not lead to an immediate impact on neutrophil activation (CD11bhigh, CD54high, CD62Lneg) or viability (DAPI+). We identified two clusters of proteins belonging to cholesterol metabolism and to the complement and coagulation cascade pathways, which are highly susceptible to neutrophil oxygen exposure during neutrophil purification. In conclusion, protecting neutrophil from oxygen during their purification and culture is recommended to avoid activation and to prevent the alteration of cytosolic protein composition., In this study, we demonstrate that preserving neutrophils from oxygen exposure during their purification and culture is recommended to avoid their activation and to prevent formation of spontaneous neutrophil extracellular traps. Studying neutrophils in vitro under controlled oxygen levels similar to those encountered in vivo is essential for a better comprehension of their physiology.

Background: Host proteases have been suggested to be crucial for dissemination of MERS, SARS-CoV, and SARS-CoV-2 coronaviruses, but the relative contribution of membrane versus intracellular proteases remains controversial. Transmembrane serine protease 2 (TMPRSS2) is regarded as one of the main proteases implicated in the coronavirus S protein priming, an important step for binding of the S protein to the angiotensin-converting enzyme 2 (ACE2) receptor before cell entry. Methods: We developed a cell-based assay to identify TMPRSS2 inhibitors. Inhibitory activity was established in SARS-CoV-2 viral load systems. Results: We identified the human extracellular serine protease inhibitor (serpin) alpha 1 anti-trypsin (A1AT) as a novel TMPRSS2 inhibitor. Structural modeling revealed that A1AT docked to an extracellular domain of TMPRSS2 in a conformation that is suitable for catalysis, resembling similar serine protease inhibitor complexes. Inhibitory activity of A1AT was established in a SARS-CoV-2 viral load system. Notably, plasma A1AT levels were associated with COVID-19 disease severity. Conclusions: Our data support the key role of extracellular serine proteases in SARS CoV-2 infections and indicate that treatment with serpins, particularly the FDA-approved drug A1AT, may be effective in limiting SARS-CoV-2 dissemination by affecting the surface of the host cells. Published version

We develop a formal model of the internal game between the leader and the factions of a party, to study the effect of party leadership on electoral success. Factions are of interest or of principle. The probability of winning an election is increasing in the leader's charisma, but also in party unity and coherence and in the factions' total contributions to party work and electoral efforts. To push factions to contribute, the leader offers both types of factions their favorite rewards in exchange for their contributions. We show that party unity and factions' total contributions are not necessarily increasing in the leader's charisma and ideological proximity to factions. Further, we show that factions of interest constraint the party's electoral strategy less than factions of principle. In particular, factions of interest always contribute more than factions of principle, are less of an obstacle towards achieving party unity, and offer the party more freedom in its choice of the ideological location and charisma of the party leader.

Neutrophils are the most abundant circulating white blood cells and are central players of the innate immune response. During their lifecycle, neutrophils mainly evolve under low oxygen conditions (0.1 - 4% O2) to which they are well adapted. Neutrophils are atypical cells since they are mainly glycolytic, and highly susceptible to oxygen-exposure, which induces their activation and death, through mechanisms which remain currently elusive. Nevertheless, nearly all studies conducted on neutrophils are carried out under atmospheric oxygen (21%), corresponding to hyperoxic conditions. Here we investigated the impact of hyperoxia during neutrophil purification and culture on neutrophil viability, activation and cytosolic protein content. Neutrophil hyper-activation (CD62L shedding) is induced during culture under hyperoxic conditions (24h), compared to neutrophils cultured under anoxic conditions. In addition, we show that maintaining neutrophils in autologous plasma is the most suitable strategy to maintain their basal state.Our results show that manipulating neutrophils under hyperoxic conditions leads to the loss of ~100 cytosolic proteins during purification, while it does not lead to an immediate impact on neutrophils activation (CD11bhigh, CD54high, CD62Llow) or viability (DAPI+). We identified two clusters of proteins belonging to the cholesterol metabolism and to the complement and coagulation cascade pathways, which are highly susceptible to neutrophil oxygen-exposure during their purification.In conclusion, preserving neutrophil from oxygen-exposure during their manipulation – purification and culture- is recommended to avoid their experimental activation and for preserving a large set of cytosolic proteins from alteration.

Shigella spp. are the causative agents of bacillary dysentery or shigellosis, mainly in children living in developing countries. The study of Shigella entire life cycle in vivo and the evaluation of vaccine candidates’ protection efficacy have been hampered by the lack of a suitable animal model of infection (1). None of the ones evaluated so far (mouse, rabbit, guinea pig) allows to recapitulate shigellosis symptoms upon Shigella oral challenge. Historical reports suggest that dysentery and scurvy are both metabolic diseases associated with ascorbate-deficiency. Mammals which are susceptible to Shigella infection (humans, non-human primates and guinea pigs) are the lonely ones which are unable to synthesize ascorbate. We optimized a low-ascorbate diet to induce moderate ascorbate-deficiency but not scurvy in guinea pigs (Ascplasma conc.=1.6 μM vs 36 μM with optimal ascorbate supply). We demonstrated that moderate ascorbate-deficiency increases shigellosis severity during extended period of time (up to 48h) with all strains tested (Shigella flexneri 5a and 2a, Shigella sonnei). At late time-points, a massive influx of neutrophils was observed both within the disrupted colonic mucosa and in the luminal compartment, although Shigella remains able to disseminate deep into the organ to reach the sub-mucosal layer and the bloodstream. This new model of shigellosis opens new doors for the study both of Shigella infection strategy and innate and adaptive immune responses to Shigella infection. It may be also of a great interest to study the virulence of other pathogen for which no suitable animal model of infection is available (Vibrio cholerae, Yersinia pestis, Mycobacterium tuberculosis or Campylobacter jejuni, among others).SignificanceThe study of Shigella virulence cycle in vivo has been hampered by the lack of a suitable animal model, which would allow the colonic mucosa infection upon oral challenge. Based on historical reports and physiological aspects, it was suggested that ascorbate-deficiency may stand as a new dysentery risk-factor. To test this hypothesis, we set up a new ascorbate-deficient guinea pig model and demonstrated for the first time that the Shigella infectious process occurred for extended period of time (up to 48h) and demonstrated that shigellosis severity was higher in ascorbate-deficient animal. Ascorbate-deficient guinea pig model of infection may be used to assess the virulence of other pathogens for which no suitable animal model of infection is still lacking.

Shigellosis results from oral ingestion of the Gram-negative bacteriaShigella, and symptoms include severe diarrhea and dysentery. In the absence of vaccines, small molecule antibacterial drugs have provided treatment options for shigellosis. However,Shigelladrug resistance is rapidly emerging, andShigellastrains with resistance to both third-generation cephalosporins and azithromycin have been identified in Asia. A re-conceptualization is needed regarding the development of therapeutics that target bacterial pathogens in order to reduce resistance development and alteration of gut microbiota, which is depleted upon treatment with wide spectrum antibiotics, thereby increasing susceptibility to subsequent enteric infections. A more organism-specific approach is to develop agents targeting virulence factors such as toxins, adhesins, invasins, quorum sensing, and protein secretion systems. ForShigella, there is interest in targeting transcription factors essential forShigellainfectionin vivorather than specific effectors. Here we describe the importance of theShigellatranscription factor IlvY inShigellavirulencein vitroandin vivo. This work included the development of a novel, oral mouse model ofShigellainfection with wild-type adult mice. Unlike previous models, mice do not require antibiotic pretreatment or diet modifications. This mouse model was used to demonstrate the importance of IlvY forShigella in vivosurvival and that deletion ofilvYimpacts host responses to infection. These results illustrate that IlvY is a potential therapeutic target for the treatment of shigellosis. In addition, the novel mouse model provides an exciting new opportunity to investigate therapeutic efficacy againstShigellainfection and host responses to infection.

Knowledge about the future response of the Greenland Ice Sheet to global climate change, including ice sheet contributions to sea level rise, is important for understanding the impact of climate change on society. Such studies rely in ice sheet model predictions and improved chronological constraints of past ice sheet extents and paleoclimatic trends. Many regions in Greenland are well studied, but northwest Greenland and especially Melville Bay, being one of the most important regions in terms of dynamical ice mass loss, lack a firm chronology of Holocene ice marginal fluctuations. In this study, we present the first comprehensive chronology for Melville Bay spanning 73.1–75.7°N based on 36 new 10Be exposure ages of boulders and 39 new radiocarbon ages of marine molluscs in Little Ice Age moraines. From weighted mean 10Be exposure ages, excluding 6 outliers, we find that the outer coast in Melville Bay was deglaciated ∼11.6 ± 0.3 ka (n = 15) and the ice margin reached its present-day position 40 km farther inland ∼11.5 ± 0.3 ka (n = 15). Our results suggest an interval of rapid ice-marginal retreat (i.e. collapse) of the northwest GrIS in Melville Bay, most likely triggered by rapidly rising atmospheric temperatures in early Holocene. Additionally, combining the comprehensive dataset of new radiocarbon ages with 26 radiocarbon ages from previous studies shows a restricted ice sheet extent from 9.1 ± 0.2 to 0.4 ± 0.1 cal ka BP, which coincides with increased sea surface temperatures. Our results highlight past ice sheet sensitivity towards climate changes in one of the least explored and most vulnerable regions of Greenland. Furthermore, comparing our new results to already existing ice sheet models (Huy3 and Huy3b) emphasize the proximal relevance of the Agassiz ice core temperature reconstruction for Melville Bay, which indicates the possible sensitivity of the ice sheet to a warming climate and place improved constraints on ice sheet simulations.

In human blood, oxygen is mainly transported by red blood cells. Accordingly, the dissolved oxygen level in plasma is expected to be limited, although it has not been quantified yet. Here, by developing dedicated methods and tools, we determined that human plasma pO2 = 8.4 mmHg (1.1% O2). Oxygen solubility in plasma was believed to be similar to water. Here we reveal that plasma has an additional ascorbate-dependent oxygen-reduction activity. Plasma experimental oxygenation oxidizes ascorbate (49.5 μM in fresh plasma vs < 2 μM in oxidized plasma) and abolishes this capacity, which is restored by ascorbate supplementation. We confirmed these results in vivo, showing that the plasma pO2 is significantly higher in ascorbate-deficient guinea pigs (Ascorbateplasma < 2 μM), compared to control (Ascorbateplasma > 15 μM). Plasma low oxygen level preserves the integrity of oxidation-sensitive components such as ubiquinol. Circulating leucocytes are well adapted to these conditions, since the abundance of their mitochondrial network is limited. These results shed a new light on the importance of oxygen exposure on leucocyte biological study, in regards with the reducing conditions they encounter in vivo; but also, on the manipulation of blood products to improve their integrity and potentially improve transfusions’ efficacy.

The roles of the inflammatory response and production of a proliferation-inducing ligand (APRIL) cytokine in gastric mucosa-associated lymphoid tissue (MALT) lymphomagenesis induced by Helicobacter species infection are not clearly understood. We characterized the gastric mucosal inflammatory response associated with gastric MALT lymphoma (GML) and identified APRIL-producing cells in two model systems: an APRIL transgenic mouse model of GML induced by Helicobacter infection (Tg-hAPRIL) and human gastric biopsy samples from Helicobacter pylori-infected GML patients. In the mouse model, polarization of T helper 1 (tbet), T helper 2 (gata3), and regulatory T cell (foxp3) responses was evaluated by quantitative PCR. In humans, a significant increase in april gene expression was observed in GML compared to gastritis. APRIL-producing cells were eosinophilic polynuclear cells located within lymphoid infiltrates, and tumoral B lymphocytes were targeted by APRIL. Together, the results of this study demonstrate that the Treg-balanced inflammatory environment is important for gastric lymphomagenesis induced by Helicobacter species, and suggest the pro-tumorigenic potential of APRIL-producing eosinophils.

In human blood, oxygen is mainly transported by red blood cells. Accordingly, the oxygen level in plasma is expected to be limited, although it has not been quantified yet. Here, by developing dedicated methods and tools, we determined that human plasma pO2 = 8.4 mmHg (1.2% O2). Oxygen solubility in plasma was believed to be similar to water. Here we reveal that plasma has an additional ascorbate-dependent oxygen-reduction activity. Plasma oxygenation oxidizes ascorbate (49.5 μM in fresh plasma vs in vivo, showing that the plasma pO2 is significantly higher in ascorbate-deficient guinea pigs (plasma ascorbate < 2μM), compared to control (plasma ascorbate > 15μM). Plasma low oxygen level preserves the integrity of oxidation-sensitive components such as ubiquinol. Circulating leucocytes are well adapted to these conditions, since the abundance of their mitochondrial network is limited.These results shed a new light on the importance of oxygen exposure on leucocyte biological study, in regards with the reducing conditions they encounter in vivo; but also on the manipulation of blood products to improve their integrity and potentially improve transfusions’ efficacy.Key points

Somatic gain-of-function mutations in isocitrate dehydrogenases (IDH) 1 and 2 are found in multiple hematologic and solid tumors, leading to accumulation of the oncometabolite (R)-2-hydroxyglutarate (2HG). 2HG competitively inhibits α-ketoglutarate–dependent dioxygenases, including histone demethylases and methylcytosine dioxygenases of the TET family, causing epigenetic dysregulation and a block in cellular differentiation. In vitro studies have provided proof of concept for mutant IDH inhibition as a therapeutic approach. We report the discovery and characterization of AG-221, an orally available, selective, potent inhibitor of the mutant IDH2 enzyme. AG-221 suppressed 2HG production and induced cellular differentiation in primary human IDH2 mutation–positive acute myeloid leukemia (AML) cells ex vivo and in xenograft mouse models. AG-221 also provided a statistically significant survival benefit in an aggressive IDH2R140Q-mutant AML xenograft mouse model. These findings supported initiation of the ongoing clinical trials of AG-221 in patients with IDH2 mutation–positive advanced hematologic malignancies. Significance: Mutations in IDH1/2 are identified in approximately 20% of patients with AML and contribute to leukemia via a block in hematopoietic cell differentiation. We have shown that the targeted inhibitor AG-221 suppresses the mutant IDH2 enzyme in multiple preclinical models and induces differentiation of malignant blasts, supporting its clinical development. Cancer Discov; 7(5); 478–93. ©2017 AACR. See related commentary by Thomas and Majeti, p. 459. See related article by Shih et al., p. 494. This article is highlighted in the In This Issue feature, p. 443

5th Congress of the European-Academy-of-Neurology (EAN), Oslo, NORWAY, JUN 29-JUL 02, 2019; International audience

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high concentrations in specific and tertiary granules of neutrophils. This protocol details how MUB40 conjugated directly to a fluorophore can be used to stain neutrophils in fixed/permeabilized tissues as well as how this can be used in live-cell imaging to assay for neutrophil activation and de-granulation. Neutrophil detection methods are limited to species-specific monoclonal antibodies, which are not always suitable for certain applications. MUB40 does not penetrate the cell membrane and is thus excluded from lactoferrin stored in non-activated/non-permeabilized neutrophils. MUB40 has the added benefit of recognizing lactoferrin from a broad host range, making it especially useful for comparing results in studies involving multiple research models, reducing the number of duplicate reagents, and simplifying protocols through single-step staining.

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high concentrations in specific and tertiary granules of neutrophils. This protocol details how MUB40 conjugated directly to a fluorophore can be used to stain neutrophils in fixed/permeabilized tissues as well as how this can be used in live-cell imaging to assay for neutrophil activation and de-granulation. Neutrophil detection methods are limited to species-specific monoclonal antibodies, which are not always suitable for certain applications. MUB40 does not penetrate the cell membrane and is thus excluded from lactoferrin stored in non-activated/non-permeabilized neutrophils. MUB40 has the added benefit of recognizing lactoferrin from a broad host range, making it especially useful for comparing results in studies involving multiple research models, reducing the number of duplicate reagents, and simplifying protocols through single-step staining.

5th Congress of the European-Academy-of-Neurology (EAN), Oslo, NORWAY, JUN 29-JUL 02, 2019; International audience

Shigella spp. are bacterial pathogens that invade the human colonic mucosa using a type III secretion apparatus (T3SA), a proteinaceous device activated upon contact with host cells. Active T3SAs translocate proteins that carve the intracellular niche of Shigella spp. Nevertheless, the activation state of the T3SA has not been addressed in vivo. Here, we used a green fluorescent protein transcription-based secretion activity reporter (TSAR) to provide a spatio-temporal description of S. flexneri T3SAs activity in the colon of Guinea pigs. First, we observed that early mucus release is triggered in the vicinity of luminal bacteria with inactive T3SA. Subsequent mucosal invasion showed bacteria with active T3SA associated with the brush border, eventually penetrating into epithelial cells. From 2 to 8 h post-challenge, the infection foci expanded, and these intracellular bacteria displayed homogeneously high-secreting activity, while extracellular foci within the lamina propria featured bacteria with low secretion activity. We also found evidence that within lamina propria macrophages, bacteria reside in vacuoles instead of accessing the cytosol. Finally, bacteria were cleared from tissues between 8 and 24 h post-challenge, highlighting the hit-and-run colonization strategy of Shigella. This study demonstrates how genetically encoded reporters can contribute to deciphering pathogenesis in vivo., We mapped Shigella flexneri cells and assessed the activity of their type III secretion apparatus in the large intestine of Guinea pigs.

Functional studies of human neutrophils and their transfusion for clinical purposes have been hampered by their short life span after isolation. Here, we demonstrate that neutrophil viability is maintained for 20 hours in culture media at 37°C under anoxic conditions with 3 mM glucose and 32 μg/mL dimethyloxalylglycine supplementation, as evidenced by stabilization of Mcl-1, proliferating cell nuclear antigen (PCNA), and pro-caspase-3. Notably, neutrophil morphology (nucleus shape and cell-surface markers) and functions (phagocytosis, degranulation, calcium release, chemotaxis, and reactive oxygen species production) were comparable to blood circulating neutrophils. The observed extension in neutrophil viability was reversed upon exposure to oxygen. Extending neutrophil life span allowed efficient transfection of plasmids (40% transfection efficiency) and short interfering RNA (interleukin-8, PCNA, and Bax), as a validation of effective and functional genetic manipulation of neutrophils both in vitro and in vivo. In vivo, transfusion of conditioned neutrophils in a neutropenic guinea pig model increased bacterial clearance of Shigella flexneri upon colonic infection, strongly suggesting that these conditioned neutrophils might be suitable for transfusion purposes. In summary, such conditioning of neutrophils in vitro should facilitate their study and offer new opportunities for genetic manipulation and therapeutic use.

The last deglaciation is the most recent interval of large-scale climate change that drove the Greenland ice sheet from continental shelf to within its present extent. Here, we use a database of 645 published 10Be ages from Greenland to document the spatial and temporal patterns of retreat of the Greenland ice sheet during the last deglaciation. Following initial retreat of its marine margins, most land-based deglaciation occurred in Greenland following the end of the Younger Dryas cold period (12.9–11.7 ka). However, deglaciation in east Greenland peaked significantly earlier (13.0–11.5 ka) than that in south Greenland (11.0–10 ka) or west Greenland (10.5–7.0 ka). The terrestrial deglaciation of east and south Greenland coincide with adjacent ocean warming. 14C ages and a recent ice-sheet model reconstruction do not capture this progression of terrestrial deglacial ages from east to west Greenland, showing deglaciation occurring later than observed in 10Be ages. This model-data misfit likely reflects the absence of realistic ice-ocean interactions. We suggest that oceanic changes may have played an important role in driving the spatial-temporal ice-retreat pattern evident in the 10Be data.

This review provides updated estimates of the glacial isostatic adjustment (GIA) component of present-day uplift at a suite of Global Navigation Satellite System (GNSS) sites in Greenland using the most recently published global ice sheet deglaciation histories. For some areas of Greenland (e.g. the north-west and north-east), the use of GNSS to estimate elastic uplift rates resulting from contemporary mass balance changes is more affected by the choice of GIA correction applied compared to other regions (e.g. central-west). The contribution of GIA to GRACE estimates of mass imbalance is becoming increasingly insignificant for large areas of Greenland as it enters a period of extreme warmth, and in total represents

The Antarctic Ice Sheet is an important indicator of climate change and driver of sea-level rise. Here we combine satellite observations of its changing volume, flow and gravitational attraction with modelling of its surface mass balance to show that it lost 2,720 ± 1,390 billion tonnes of ice between 1992 and 2017, which corresponds to an increase in mean sea level of 7.6 ± 3.9 millimetres (errors are one standard deviation). Over this period, ocean-driven melting has caused rates of ice loss from West Antarctica to increase from 53 ± 29 billion to 159 ± 26 billion tonnes per year; ice-shelf collapse has increased the rate of ice loss from the Antarctic Peninsula from 7 ± 13 billion to 33 ± 16 billion tonnes per year. We find large variations in and among model estimates of surface mass balance and glacial isostatic adjustment for East Antarctica, with its average rate of mass gain over the period 1992–2017 (5 ± 46 billion tonnes per year) being the least certain.

Neutrophils represent the most abundant immune cells recruited to inflamed tissues. A lack of dedicated tools has hampered their detection and study. We show that a synthesized peptide, MUB40, binds to lactoferrin, the most abundant protein stored in neutrophil-specific and tertiary granules. Lactoferrin is specifically produced by neutrophils among other leukocytes, making MUB40 a specific neutrophil marker. Naive mammalian neutrophils (human, guinea pig, mouse, rabbit) were labeled by fluorescent MUB40 conjugates (-Cy5, Dylight405). A peptidase-resistant retro-inverso MUB40 (RI-MUB40) was synthesized and its lactoferrin-binding property validated. Neutrophil lactoferrin secretion during in vitro Shigella infection was assessed with RI-MUB40-Cy5 using live cell microscopy. Systemically administered RI-MUB40-Cy5 accumulated at sites of inflammation in a mouse arthritis inflammation model in vivo and showed usefulness as a potential tool for inflammation detection using non-invasive imaging. Improving neutrophil detection with the universal and specific MUB40 marker will aid the study of broad ranges of inflammatory diseases. MUB40 is a universal and specific marker of mammalian neutrophils. MUB40 interacts with lactoferrin, stored in specific (β1), tertiary (β2), and secretome (γ) granules secreted upon stimulation. Neutrophil detection in inflamed or infected tissues is facilitated by MUB40 peptides conjugated to fluorophores. Neutrophilic inflammation detection may be envisaged by combining MUB40 with non-invasive imaging.

Neutrophils represent the most abundant immune cells recruited to inflamed tissues. A lack of dedicated tools has hampered their detection and study. We show that a synthesized peptide, MUB

Recurrent mutations at R140 and R172 in isocitrate dehydrogenase 2 (IDH2) occur in many cancers, including ∼12% of acute myeloid leukemia (AML). In preclinical models these mutations cause accumulation of the oncogenic metabolite R-2-hydroxyglutarate (2-HG) and induce hematopoietic differentiation block. Single-agent enasidenib (AG-221/CC-90007), a selective mutant IDH2 (mIDH2) inhibitor, produced an overall response rate of 40.3% in relapsed/refractory AML (rrAML) patients with mIDH2 in a phase 1 trial. However, its mechanism of action and biomarkers associated with response remain unclear. Here, we measured 2-HG, mIDH2 allele burden, and co-occurring somatic mutations in sequential patient samples from the clinical trial and correlated these with clinical response. Furthermore, we used flow cytometry to assess inhibition of mIDH2 on hematopoietic differentiation. We observed potent 2-HG suppression in both R140 and R172 mIDH2 AML subtypes, with different kinetics, which preceded clinical response. Suppression of 2-HG alone did not predict response, because most nonresponding patients also exhibited 2-HG suppression. Complete remission (CR) with persistence of mIDH2 and normalization of hematopoietic stem and progenitor compartments with emergence of functional mIDH2 neutrophils were observed. In a subset of CR patients, mIDH2 allele burden was reduced and remained undetectable with response. Co-occurring mutations in NRAS and other MAPK pathway effectors were enriched in nonresponding patients, consistent with RAS signaling contributing to primary therapeutic resistance. Together, these data support differentiation as the main mechanism of enasidenib efficacy in relapsed/refractory AML patients and provide insight into resistance mechanisms to inform future mechanism-based combination treatment studies.

In this study the Holocene evolution of Hans Tausen Iskappe (Peary Land, North Greenland) is investigated. Constraints on the ice cap evolution are combined with climatic records in a numerical ice flow – surface mass balance (SMB) model to better understand the palaeoenvironmental and climatic evolution of this region. Our simulations suggest that after disconnecting from the Greenland Ice Sheet (GrIS) the ice cap had roughly its present-day size and geometry around 9–8.5 ka BP. During the Holocene Thermal Maximum (HTM) the southern part of the ice cap is modelled to collapse, while the northern part of the ice cap survived this warmer period. The late Holocene regrowth of the ice cap to its maximum Neoglacial extent at the end of the Little Ice Age (LIA) can be reproduced from the temperature reconstruction. The simulations suggest that over the last millennia the local precipitation may have been up to 70–80% higher than at present. By coupling the pre-industrial temperature forcing to a post-LIA warming trend, it is suggested that the warming between the end of the LIA and the period 1961–1990 was between 1 and 2 °C. In all experiments the ice flow model complexity and horizontal resolution have only a minor effect on the long-term evolution of the ice cap. We further conclude that the glacial isostatic adjustment has a significant effect on the modelled Holocene ice cap evolution. This suggests that modelling studies of millennial-scale ice cap evolution should focus on SMB and boundary conditions, rather than on complex ice dynamics.

Shigella is a leading cause of dysentery worldwide, with the majority of infections caused by two subgroups, S. flexneri and S. sonnei. Although S. flexneri has been highly prevalent in low-income countries, global development has brought an increase in S. sonnei at the expense of S. flexneri. However, the mechanisms behind this shift are not understood. Here we report that S. sonnei, but not S. flexneri, encodes a type VI secretion system (T6SS) that provides a competitive advantage in the gut. S. sonnei competes against E. coli and S. flexneri in mixed cultures, but this advantage is reduced in T6SS mutant strains. In addition, S. sonnei can persist as well as outcompete E. coli and S. flexneri in mice in a T6SS-dependent manner. These findings suggest that S. sonnei has a competitive advantage over S. flexneri and potentially explain the increasing global prevalence of S. sonnei.

We present a revised and extended high Arctic air temperature reconstruction from a single proxy that spans the past ∼12,000 y (up to 2009 CE). Our reconstruction from the Agassiz ice cap (Ellesmere Island, Canada) indicates an earlier and warmer Holocene thermal maximum with early Holocene temperatures that are 4–5 °C warmer compared with a previous reconstruction, and regularly exceed contemporary values for a period of ∼3,000 y. Our results show that air temperatures in this region are now at their warmest in the past 6,800–7,800 y, and that the recent rate of temperature change is unprecedented over the entire Holocene. The warmer early Holocene inferred from the Agassiz ice core leads to an estimated ∼1 km of ice thinning in northwest Greenland during the early Holocene using the Camp Century ice core. Ice modeling results show that this large thinning is consistent with our air temperature reconstruction. The modeling results also demonstrate the broader significance of the enhanced warming, with a retreat of the northern ice margin behind its present position in the mid Holocene and a ∼25% increase in total Greenland ice sheet mass loss (∼1.4 m sea-level equivalent) during the last deglaciation, both of which have implications for interpreting geodetic measurements of land uplift and gravity changes in northern Greenland.

A contribution to the PAGES' PALSEA2 Working Group. ABSTRACT: This paper presents new Holocene relative sea level (RSL) data collected from isolation basins close to the town of Paamiut in south west Greenland. The data shows a rapid fall from a marine limit of c. 52masl at c. 10.9cal.ka BP to close to present by c. 9.5cal.ka BP at rates of up to c. 32mm/yr falling below present for the majority of the Holocene before rising to present in the last 2000 years. The elevation of the RSL lowstand is not well constrained but was at least below 3m. This pattern of rapid RSL fall during the early Holocene matches the pattern seen at other southern Greenland locations suggesting rapid largely simultaneous ice retreat from the area surrounding the Qassimiut Lobe at the start of the Holocene occurring c. 2000 years after the initial deglaciation of the extreme southern tip of Greenland. The RSL histories from this and other southern Greenland locations are distinct to those recorded further north along the west coast and are in broad agreement with a pattern of vertical land motion and RSL predicted by the Huy2 model (Simpson etal. 2009) which predicts an 80m drop in the contribution of vertical land motion to RSL at 10cal.ka BP between Sisimiut and Paamiut on the west coast. Despite this broad scale spatial agreement between the RSL data and the Huy2 model it fails to satisfactorily predict the Holocene RSL histories at Paamiut and other southern Greenland locations. Sensitivity tests indicate that the data model misfits are most likely due to an over estimate of the forcing during the Holocene Thermal Maximum (or the response to this forcing) in southern Greenland and error in the North American ice sheets component of the background deglaciation model. Our new data suggests that much of the southern part of the ice sheet acted differently to the area further north. However RSL changes at Paamiut are also largely impacted by regional and larger scale processes including a bulls eye of uplift centred on the west the impact of the Holocene Thermal Maximum and the influence of the collapse of the North American ice sheets. © 2013 Elsevier Ltd.

Erratum in Bioengineered Human Organ-on-Chip Reveals Intestinal Microenvironment and Mechanical Forces Impacting Shigella Infection. [Cell Host Microbe. 2019]; International audience; Intestinal epithelial cells are constantly exposed to pathogens and mechanical forces. However, the impact of mechanical forces on infections leading to diarrheal diseases remains largely unknown. Here, we addressed whether flow and peristalsis impact the infectivity of the human pathogen Shigella within a 3D colonic epithelium using Intestine-Chip technology. Strikingly, infection is significantly increased and minimal bacterial loads are sufficient to invade enterocytes from the apical side and trigger loss of barrier integrity, thereby shifting the paradigm about early stage Shigella invasion. Shigella quickly colonizes epithelial crypt-like invaginations and demonstrates the essential role of the microenvironment. Furthermore, by modulating the mechanical forces of the microenvironment, we find that peristalsis impacts Shigella invasion. Collectively, our results reveal that Shigella leverages the intestinal microenvironment by taking advantage of the microarchitecture and mechanical forces to efficiently invade the intestine. This approach will enable molecular and mechanistic interrogation of human-restricted enteric pathogens.

Ice core records were recently used to infer elevation changes of the Greenland ice sheet throughout the Holocene. The inferred elevation changes show a significantly greater elevation reduction than those output from numerical models, bringing into question the accuracy of the model-based reconstructions and, to some extent, the estimated elevation histories. A key component of the ice core analysis involved removing the influence of vertical surface motion on the δ 18 O signal measured from the Agassiz and Renland ice caps. We re-visit the original analysis with the intent to determine if the use of more accurate land uplift curves can account for some of the above noted discrepancy. To improve on the original analysis, we apply a geophysical model of glacial isostatic adjustment calibrated to sea-level records from the Queen Elizabeth Islands and Greenland to calculate the influence of land height changes on the δ 18 O signal from the two ice cores. This procedure is complicated by the fact that δ 18 O contained in Agassiz ice is influenced by land height changes distant from the ice cap and so selecting a single location at which to compute the land height signal is not possible. Uncertainty in this selection is further complicated by the possible influence of Innuitian ice during the early Holocene (12–8 ka BP). Our results indicate that a more accurate treatment of the uplift correction leads to elevation histories that are, in general, shifted down relative to the original curves at GRIP, NGRIP, DYE-3 and Camp Century. In addition, compared to the original analysis, the 1-σ uncertainty is considerably larger at GRIP and NGRIP. These changes reduce the data-model discrepancy reported by Vinther et al. (2009) at GRIP, NGRIP, DYE-3 and Camp Century. A more accurate treatment of isostasy and surface loading also acts to improve the data-model fits such that the residuals at all four sites for the period 8 ka BP to present are significantly reduced compared to the original analysis. Prior to 8 ka BP, the possible influence of Innuitian ice on the inferred elevation histories prevents a meaningful comparison.

Shigella is a pathovar of Escherichia coli comprising 4 groups, Shigella flexneri, Shigella sonnei, Shigella dysenteriae, and Shigella boydii, each of them, with the exception of S. sonnei, comprising several serotypes. Shigella accounts for the majority of dysentery causing infections occurring world-wide each year. Recent advancements in the Shigella field have led to a better understanding of the molecular mechanisms underlying host epithelial cell invasion and immune cell function manipulation, mainly using S. flexneri as a model. Host-cell invasion is the final step of the infection process, as Shigella virulence strategy relies also on its ability to survive hostile conditions during its journey through the gastro-intestinal tract, to compete with the host microbiota and to cross the intestinal mucus layer. Hence, the diversity of the virulence strategies among the different Shigella species has not yet been deeply investigated, which might be an important step to understand the epidemiological spreading of Shigella species worldwide and a key aspect for the validation of novel vaccine candidates. The recent development of high Throughput screening and sequencing methods will facilitate these complex comparison studies.In this review we discuss several of the major avenues that the Shigella research field has taken over the past few years and hopefully gain some insights into the questions that remain surrounding this important human pathogen.

Shigella is a pathovar of Escherichia coli comprising four groups, Shigella flexneri, Shigella sonnei, Shigella dysenteriae, and Shigella boydii, each of them, with the exception of S.sonnei, comprising several serotypes. Shigella accounts for the majority of dysentery causing infections occurring world-wide each year. Recent advancements in the Shigella field have led to a better understanding of the molecular mechanisms underlying host epithelial cell invasion and immune cell function manipulation, mainly using S. flexneri as a model. Host-cell invasion is the final step of the infection process, as Shigella's virulence strategy relies also on its ability to survive hostile conditions during its journey through the gastro-intestinal tract, to compete with the host microbiota and to cross the intestinal mucus layer. Hence, the diversity of the virulence strategies among the different Shigella species has not yet been deeply investigated, which might be an important step to understand the epidemiological spreading of Shigella species worldwide and a key aspect for the validation of novel vaccine candidates. The recent development of high-throughput screening and sequencing methods will facilitate these complex comparison studies. In this review we discuss several of the major avenues that the Shigella research field has taken over the past few years and hopefully gain some insights into the questions that remain surrounding this important human pathogen.

EA and J-YT contributed equally to this work; International audience; Hypoxia is defined as a tissue oxygenation status below physiological needs. During Shigella infection, an infectious hypoxia is induced within foci of infection. In this review, we discuss how Shigella physiology and virulence are modulated and how the main recruited immune cells, the neutrophils, adapt to this environment.

Objective: To test the a priori hypothesis that longer duration of ruptured membranes is associated with increased risk of vertical transmission of HIV. Design: The relationship between duration of ruptured membranes and vertical transmission of HIV was evaluated in an individual patient data meta-analysis. Methods: Eligible studies were prospective cohort studies including at least 100 mother-child pairs, from regions where HIV-infected women are counselled not to breastfeed. Analyses were restricted to vaginal deliveries and non-elective Cesarean sections; elective Cesarean section deliveries (those performed before onset of labour and before rupture of membranes) were excluded. Results: The primary analysis included 4721 deliveries with duration of ruptured membranes ≤ 24 h. After adjusting for other factors known to be associated with vertical transmission using logistic regression analysis to assess the strength of the relationship, the risk of vertical HIV transmission increased approximately 2% with an increase of 1 h in the duration of ruptured membranes [adjusted odds ratio, 1.02; 95% confidence interval, 1.01-1.04; for each 1 h increment]. There were no significant interactions of duration of ruptured membranes with study cohort or with any of the covariates, except maternal AIDS. Among women diagnosed with AIDS, the estimated probability of transmission increased from 8% to 31% with duration of ruptured membranes of 2 h and 24 h respectively (P < 0.01). Conclusions: These results support the importance of duration of ruptured membranes as a risk factor for vertical transmission of HIV and suggest that a diagnosis of AIDS in the mother at the time of delivery may potentiate the effect of duration of ruptured membranes. © 2001 Lippincott Williams & Wilkins.

Intestinal pathogens use the host's excessive inflammatory cytokine response, designed to eliminate dangerous bacteria, to disrupt epithelial gut wall integrity and promote their tissue invasion. We sought to develop a non-antibiotic-based approach to prevent this injury. Molecular docking studies suggested that glycosylated dendrimers block the TLR4-MD-2-LPS complex, and a 13.6 kDa polyamidoamine (PAMAM) dendrimer glucosamine (DG) reduced the induction of human monocyte interleukin (IL)-6 by Gram-negative bacteria. In a rabbit model of shigellosis, PAMAM-DG prevented epithelial gut wall damage and intestinal villous destruction, reduced local IL-6 and IL-8 expression, and minimized bacterial invasion. Computational modelling studies identified a 3.3 kDa polypropyletherimine (PETIM)-DG as the smallest likely bioactive molecule. In human monocytes, high purity PETIM-DG potently inhibited Shigella Lipid A-induced IL-6 expression. In rabbits, PETIM-DG prevented Shigella-induced epithelial gut wall damage, reduced local IL-6 and IL-8 expression, and minimized bacterial invasion. There was no change in β-defensin, IL-10, interferon-β, transforming growth factor-β, CD3 or FoxP3 expression. Small and orally delivered DG could be useful for preventing gut wall tissue damage in a wide spectrum of infectious diarrhoeal diseases. –>See accompanying article http://dx.doi.org/10.1002/emmm.201201668

The gastrointestinal tract provides a variety of environmental challenges to any bacterium seeking to successfully colonize or cause disease in a host. A major obstacle is the varied oxygen concentrations encountered at different sites in the intestine. Here we review the mechanisms bacterial pathogens utilize to sense oxygen within the gastrointestinal tract, and recent insights into how this acts as a signal to trigger virulence and to modulate host responses.

Background Enteric glial cells (EGCs) are important regulators of intestinal epithelial barrier (IEB) functions. EGC-derived S-nitrosoglutathione (GSNO) has been shown to regulate IEB permeability. Whether EGCs and GSNO protect the IEB during infectious insult by pathogens such as Shigella flexneri is not known. Methods S flexneri effects were characterised using in vitro coculture models of Caco-2 cells and EGCs (or GSNO), ex vivo human colonic mucosa, and in vivo ligated rabbit intestinal loops. The effect of EGCs on S flexneri-induced changes in the invasion area and the inflammatory response were analysed by combining immunohistochemical, ELISA and PCR methods. Expression of small G-proteins was analysed by western blot. Expression of ZO-1 and localisation of bacteria were analysed by fluorescence microscopy. Results EGCs significantly reduced barrier lesions and inflammatory response induced by S flexneri in Caco-2 monolayers. The EGC-mediated effects were reproduced by GSNO, but not by reduced glutathione, and pharmacological inhibition of pathways involved in GSNO synthesis reduced EGC protecting effects. Furthermore, expression of Cdc42 and phospho-PAK in Caco-2 monolayers was significantly reduced in the presence of EGCs or GSNO. In addition, changes in ZO-1 expression and distribution induced by S flexneri were prevented by EGCs and GSNO. Finally, GSNO reduced S flexneriinduced lesions of the IEB in human mucosal colonic explants and in a rabbit model of shigellosis. Conclusion These results highlight a major protective function of EGCs and GSNO in the IEB against S flexneri attack. Consequently, this study lays the scientific basis for using GSNO to reduce barrier susceptibility to infectious or inflammatory challenge.