Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Distribution, Diversity and Roles of CRISPR-Cas Systems in Human and Animal Pathogenic Streptococci

International audienceStreptococci form a wide group of bacteria and are involved in both human and animal pathologies. Among pathogenic isolates, differences have been highlighted especially concerning their adaptation and virulence profiles. CRISPR-Cas systems have been identified in bacteria and many streptococci harbor one or more systems, particularly subtypes I-C, II-A, and III-A. Since the demonstration that CRISPR-Cas act as an adaptive immune system in Streptococcus thermophilus , a lactic bacteria, the diversity and role of CRISPR-Cas were extended to many germs and functions were enlarged. Among those, the genome editing tool based on the properties of Cas endonucleases is used worldwide, and the recent attribution of the Nobel Prize illustrates the importance of this tool in the scientific world. Another application is CRISPR loci analysis, which allows to easily characterize isolates in order to understand the interactions of bacteria with their environment and visualize species evolution. In this review, we focused on the distribution, diversity and roles of CRISPR-Cas systems in the main pathogenic streptococci

Group B Streptococcus CRISPR1 Typing of Maternal, Fetal, and Neonatal Infectious Disease Isolates Highlights the Importance of CC1 in In Utero Fetal Death

ABSTRACT We performed a descriptive analysis of group B Streptococcus (GBS) isolates responsible for maternal and fetal infectious diseases from 2004 to 2020 at the University Hospital of Tours, France. This represents 115 isolates, including 35 isolates responsible for early-onset disease (EOD), 48 isolates responsible for late-onset disease (LOD), and 32 isolates from maternal infections. Among the 32 isolates associated with maternal infection, 9 were isolated in the context of chorioamnionitis associated with in utero fetal death. Analysis of neonatal infection distribution over time highlighted the decrease in EOD since the early 2000s, while LOD incidence has remained relatively stable. All GBS isolates were analyzed by sequencing their CRISPR1 locus, which is an efficient way to determine the phylogenetic affiliation of strains, as it correlates with the lineages defined by multilocus sequence typing (MLST). Thus, the CRISPR1 typing method allowed us to assign a clonal complex (CC) to all isolates; among these isolates, CC17 was predominant (60/115, 52%), and the other main CCs, such as CC1 (19/115, 17%), CC10 (9/115, 8%), CC19 (8/115, 7%), and CC23 (15/115, 13%), were also identified. As expected, CC17 isolates (39/48, 81.3%) represented the majority of LOD isolates. Unexpectedly, we found mainly CC1 isolates (6/9) and no CC17 isolates that were responsible for in utero fetal death. Such a result highlights the possibility of a particular role of this CC in in utero infection, and further investigations should be conducted on a larger group of GBS isolated in a context of in utero fetal death. IMPORTANCE Group B Streptococcus is the leading bacterium responsible for maternal and neonatal infections worldwide, also involved in preterm birth, stillbirth, and fetal death. In this study, we determined the clonal complex of all GBS isolates responsible for neonatal diseases (early- and late-onset diseases) and maternal invasive infections, including chorioamnionitis associated with in utero fetal death. All GBS was isolated at the University Hospital of Tours from 2004 to 2020. We described the local group B Streptococcus epidemiology, which confirmed national and international data concerning neonatal disease incidence and clonal complex distribution. Indeed, neonatal diseases are mainly characterized by CC17 isolates, especially in late-onset disease. Interestingly, we identified mainly CC1 isolates responsible for in utero fetal death. CC1 could have a particular role in this context, and such a result should be confirmed on a larger group of GBS isolated from in utero fetal death

Aspects logistiques de la transfusion de concentrés érythrocytaires en milieu préhospitalier

Pre-hospital red blood cell transfusion is already used in many countries, both in military and civilian settings, and provides a better chance of survival for patients suffering from massive bleeding. However, this is not a current practice in Switzerland. This article aims to study Swiss specificities and provide a turnkey concept for the implementation of red blood cell transfusion in an emergency pre-hospital setting, by road or by air. The transfusion benefits and risks, the logistical aspect and the costs are discussed.La transfusion de concentrés érythrocytaires (CE) en milieu préhospitalier est déjà réalisée dans de nombreux pays tant dans un contexte militaire que civil et permet d’augmenter les chances de survie des patients souffrant d’hémorragie massive. En Suisse, cette pratique n’est pas courante. Cet article a pour but d’étudier les spécificités suisses et de proposer un concept clé en main pour l’implémentation de la transfusion de CE dans un service de sauvetage médicalisé terrestre ou héliporté. Les bénéfices et les risques de la transfusion, les modalités logistiques et les coûts y sont abordés

Long-term surveillance of group B Streptococcus strains isolated from infection and colonization in pregnant women and newborns

International audienceIntroduction. Group B Streptococcus (GBS) remains the leading cause of bacterial neonatal infections worldwide, despite the spread of recommendations on vaginal screening and antibiotic prophylaxis. Hypothesis/Gap Statement. There is a need to evaluate the potential changes in GBS epidemiology over time following the introduction of such guidelines. Aim. Our aim was to perform a descriptive analysis of the epidemiological characteristics of GBS by conducting a long-term surveillance of strains isolated between 2000 and 2018, using molecular typing methods. Methodology. A total of 121 invasive strains, responsible for maternal infections (20 strains), fetal infections (8 strains) and neonatal infections (93 strains), were included in the study, representing all the invasive isolates during the period; in addition, 384 colonization strains isolated from vaginal or newborn samples were randomly selected. The 505 strains were characterized by capsular polysaccharide (CPS) type multiplex PCR assay and the clonal complex (CC) was assigned using a single nucleotide polymorphism PCR assay. Antibiotic susceptibility was also determined. Results. CPS types III (32.1 % of the strains), Ia (24.6 %) and V (19 %) were the most prevalent. The five main CCs observed were CC1 (26.3 % of the strains), CC17 (22.2 %), CC19 (16.2 %), CC23 (15.8 %) and CC10 (13.9 %). Neonatal invasive GBS diseases were predominantly due to CC17 isolates (46.3 % of the strains), which mainly express CPS type III (87.5 %), with a very high prevalence in late-onset diseases (76.2 %). Conclusion. Between 2000 and 2018, we observed a decrease in the proportion of CC1 strains, which mainly express CPS type V, and an increase in the proportion of CC23 strains, mainly expressing CPS type Ia. Conversely, there was no significant change in the proportion of strains resistant to macrolides, lincosamides or tetracyclines. The two molecular techniques used in our study provide almost as much information as classical serotyping and multilocus sequence typing, but are quicker, easy to perform, and avoid long sequencing and analysis steps

Soil Eukaryotic Diversity: A Metatranscriptomic Approach

International audienc

Cellular accumulation of fluoroquinolones is not predictive of their intracellular activity studies with gemifloxacin, moxifloxacin and ciprofloxacin in a pharmacokinetic/pharmacodynamic model of uninfected and infected macrophages

Fluoroquinolones enter eukaryotic cells but the correlation between cellular accumulation and activity remains poorly established. Gemifloxacin is known to accumulate to a larger extent than most other fluoroquinolones in tissues. Using murine J774 macrophages and human THP-1 monocytes, we show that gemifloxacin accumulates more than ciprofloxacin and even moxifloxacin. Whilst showing indistinguishable kinetics of accumulation in J774 macrophages, gemifloxacin was released at an approximately two-fold slower rate than ciprofloxacin and its release was only partial. Gemifloxacin was also a weaker substrate than ciprofloxacin for the efflux transporter Mrp4 active in J774 macrophages. In cells infected with Listeria monocytogenes or Staphylococcus aureus (typical cytoplasmic and phagolysosomal organisms, respectively), gemifloxacin was equipotent to moxifloxacin and ciprofloxacin in concentration-dependent experiments if data are normalised based on the minimum inhibitory concentration (MIC) in broth. Thus, larger cellular concentrations of gemifloxacin than of moxifloxacin or ciprofloxacin were needed to obtain a similar target effect. Fractionation studies showed a similar subcellular distribution for all three fluoroquinolones, with approximately two-thirds of the cell-associated drug recovered in the soluble fraction (cytosol). These data suggest that cellular accumulation of fluoroquinolones is largely a self-defeating process as far as activity is concerned, with the intracellular drug made inactive in proportion to its accumulation level. Whilst these observations do not decrease the intrinsic value of fluoroquinolones for the treatment of intracellular infections, they indicate that ranking fluoroquinolones based on cell accumulation data without measuring the corresponding intracellular activity may lead to incorrect conclusions regarding their real potential

Vimentin expression predicts the occurrence of metastases in non small cell lung carcinomas.

Epithelial-to-mesenchymal transition (EMT) is believed to contribute to tumour invasion. Vimentin expression by carcinoma cells is a largely recognized marker of EMT. This study aimed at examining vimentin expression in non small cell lung carcinomas (NSCLC) by immunohistochemistry to evaluate potential correlations between vimentin expression and the differentiation status, the TNM stage and the outcome of the patients. 295 NSCLC including 164 squamous cell carcinomas (SCC), 108 adenocarcinomas (AC) and 23 other NSCLC carcinomas have been examined by immunohistochemistry. Vimentin was indeed detected in 145 cases (49.2%). It was principally present in isolated tumour cells and invasive clusters, particularly in cells at the tumour/stroma interface. Vimentin expression was significantly more expressed in large cell neuroendocrine, adeno-squamous and sarcomatoid carcinomas than in SCC and AC and was significantly associated with the differentiation status of carcinomas. The follow-up of 193 patients further demonstrated that an extensive expression of vimentin (>50% of tumour cells) was associated with the occurrence of metastases. In conclusion, our data demonstrate that vimentin expression is a frequent event in NSCLC and that its expression can be associated with a lack of differentiation and the occurrence of metastases

Cellular accumulation of fluoroquinolones is not predictive of their intracellular activity: Studies with gemifloxacin, moxifloxacin and ciprofloxacin in a pharmacokinetic/pharmacodynamic model of uninfected and infected macrophages

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ijantimicag.2011.05.011Fluoroquinolones enter eukaryotic cells but the correlation between cellular accumulation and activity remains poorly established. Gemifloxacin is known to accumulate to a larger extent than most other fluoroquinolones in tissues. Using murine J774 macrophages and human THP-1 monocytes, we show that gemifloxacin accumulates more than ciprofloxacin and even moxifloxacin. Whilst showing indistinguishable kinetics of accumulation in J774 macrophages, gemifloxacin was released at an approximately two-fold slower rate than ciprofloxacin and its release was only partial. Gemifloxacin was also a weaker substrate than ciprofloxacin for the efflux transporter Mrp4 active in J774 macrophages. In cells infected with Listeria monocytogenes or Staphylococcus aureus (typical cytoplasmic and phagolysosomal organisms, respectively), gemifloxacin was equipotent to moxifloxacin and ciprofloxacin in concentration-dependent experiments if data are normalised based on the minimum inhibitory concentration (MIC) in broth. Thus, larger cellular concentrations of gemifloxacin than of moxifloxacin or ciprofloxacin were needed to obtain a similar target effect. Fractionation studies showed a similar subcellular distribution for all three fluoroquinolones, with approximately two-thirds of the cell-associated drug recovered in the soluble fraction (cytosol). These data suggest that cellular accumulation of fluoroquinolones is largely a self-defeating process as far as activity is concerned, with the intracellular drug made inactive in proportion to its accumulation level. Whilst these observations do not decrease the intrinsic value of fluoroquinolones for the treatment of intracellular infections, they indicate that ranking fluoroquinolones based on cell accumulation data without measuring the corresponding intracellular activity may lead to incorrect conclusions regarding their real potential. © 2011 Elsevier B.V. and the International Society of Chemotherapy.CMV is Boursier of the Belgian Fonds pour la Recherche dans l’Industrie et l’Agriculture (FRIA); BM was postdoctoral fellow of the First post-doctoral programme of the Belgian Région wallonne; SL and FVB are Chargé de Recherches and Maître de Recherches, respectively, of the Belgian Fonds de la Recherche Scientifique (F.R.S.-FNRS). This work was supported by the Belgian Fonds de la Recherche Scientifique Médicale (grants no. 3.4.597.06 and 3.4.583.08) and the Belgian Fonds de la Recherche Scientifique (grant no. 1.5.195.07).Peer Reviewe