Extracellular Vesicles: Translational Agenda Questions for Three Protozoan Parasites.

The protozoan parasites Plasmodium falciparum, Leishmania spp. and Trypanosoma cruzi continue to exert a significant toll on the disease landscape of the human population in sub-Saharan Africa and Latin America. Control measures have helped reduce the burden of their respective diseases-malaria, leishmaniasis and Chagas disease-in endemic regions. However, the need for new drugs, innovative vaccination strategies and molecular markers of disease severity and outcomes has emerged because of developing antimicrobial drug resistance, comparatively inadequate or absent vaccines, and a lack of trustworthy markers of morbid outcomes. Extracellular vesicles (EVs) have been widely reported to play a role in the biology and pathogenicity of P. falciparum, Leishmania spp. and T. cruzi ever since they were discovered. EVs are secreted by a yet to be fully understood mechanism in protozoans into the extracellular milieu and carry a cargo of diverse molecules that reflect the originator cell's metabolic state. Although our understanding of the biogenesis and function of EVs continues to deepen, the question of how EVs in P. falciparum, Leishmania spp. and T. cruzi can serve as targets for a translational agenda into clinical and public health interventions is yet to be fully explored. Here, as a consortium of protozoan researchers, we outline a plan for future researchers and pose three questions to direct an EV's translational agenda in P. falciparum, Leishmania spp. and T. cruzi. We opine that in the long term, executing this blueprint will help bridge the current unmet needs of these medically important protozoan diseases in sub-Saharan Africa and Latin America.

Ecological determinants of leishmaniasis vector, Lutzomyia spp.: A scoping review.

Leishmania spp. are zoonotic parasites transmitted by phlebotomine sand flies, including those of the Lutzomyia genus, which can cause leishmaniases in both humans and dogs. Lutzomyia spp. are established in many countries in South and Central America and some areas of the southern United States, with suspected potential of these vectors to undergo further range expansion due to climate change. A scoping review was conducted following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extensions for Scoping Reviews (PRISMA-ScR) guidelines to describe the current state of knowledge on the key ecological factors associated with Lutzomyia spp. survival, reproduction and establishment. The following electronic databases were searched for eligible studies published from 1 January 1990, to the date of search, 26 April 2023: CAB Direct (CABI), MEDLINE (via Ovid), Biological Sciences Database and Environmental Sciences Database. Primary research articles that were available in English and focused on ecological factors associated with Lutzomyia spp., such as climatic and habitat factors, geographic range, seasonality and temporality, and host abundance, were eligible for inclusion in the study. Following de-duplication, a total of 167 studies were included in Level 1 screening, 64 studies were included in Level 2 screening and 31 studies met the criteria for data extraction. Study locations included Argentina, Brazil, Colombia, Peru, Venezuela, the United States, Mexico and Canada, with some studies including multiple regions. A total of 31 different Lutzomyia spp. were assessed across these studies, with most (51.6%) of the studies focused on Lutzomyia longipalpis. Eligible studies investigated factors such as seasonality (n = 5), temperature (n = 19), precipitation (n = 13), humidity (n = 2), vegetation presence or requirements (n = 13), ecotypes (n = 7), and/or community type (i.e., urban, suburban, rural) (n = 5). Lutzomyia spp. activity was found to be higher during the rainy season, and peak when temperatures were between 20 and 25°C. Lutzomyia spp. were also found to preferentially reside in tropical or subtropical forests, which are characterised by their lack of a distinct dry season and high precipitation. This scoping review summarised the current state of the literature on the ecological factors associated with the survival, activity and reproduction of Lutzomyia spp. While there appears to be some consensus in the literature regarding some ecological requirements (such as seasonality, temperature and habitat features), overall, there is a lack of published research in this topic. This poses a significant challenge for future studies, which aim to predict the future distribution of Lutzomyia spp. in the context of climate and land use changes. Additional ecological research is urgently needed on Lutzomyia spp. given their relevance to both human and animal health.

EVALUATION OF CLINICAL DIAGNOSTICS FOR PROVENTRICULAR NEMATODIASIS DUE TO SYNHIMANTUS NASUTA IN LORIKEETS (TRICHOGLOSSUS SPP.).

In this case series, clinical investigations were pursued during a Synhimantus nasuta infection in a lorikeet (Trichoglossus spp.) flock outbreak situation to better describe and document clinical presentations. In 11 lorikeets suspected to be infected with Synhimantus based on at least one abnormal finding on their physical examination (lethargy, feather-damaging behavior on the ventrum, weight loss, pale iris), the presence of five additional parameters was documented: anemia, relative eosinophilia, increased proventricular diameter-to-keel height ratio (PKR), proventricular barium filling defect, and positive fecal occult blood detection test. A total score (X of 9) was calculated by combining all these findings. Synhimantus nasuta infection was confirmed in four of these individuals by modified Wisconsin fecal examination. Suspected cases (n = 7 of 11) presented only with low scores (1-3 of 9), whereas birds with confirmed infections (n = 4 of 11) presented with both low (1-3 of 9, n = 2 of 4) and high (6-7 of 9, n = 2 of 4) total scores. High scores were associated with clinical anemia. Fecal occult blood was present in all confirmed cases and 4 of 7 suspected cases. An enlarged proventriculus was only observed in birds with active shedding (n = 3 of 4). Follow-up evaluations after 6 mon of treatment with ivermectin and selamectin suggested complete recovery with lowered or normalized total scores. In conclusion, during an S. nasuta outbreak, a rapid physical examination helps to identify suspect cases, including individuals requiring immediate medical attention. In the absence of ova shedding, infection cannot be excluded on the basis of scarce clinical findings, but the detection of occult fecal blood and an increased PKR should raise the index of suspicion.

Influence of N-Methylation and Conformation on Almiramide Anti-Leishmanial Activity.

The almiramide N-methylated lipopeptides exhibit promising activity against trypanosomatid parasites. A structure-activity relationship study has been performed to examine the influences of N-methylation and conformation on activity against various strains of leishmaniasis protozoan and on cytotoxicity. The synthesis and biological analysis of twenty-five analogs demonstrated that derivatives with a single methyl group on either the first or fifth residue amide nitrogen exhibited greater activity than the permethylated peptides and relatively high potency against resistant strains. Replacement of amino amide residues in the peptide, by turn inducing α amino γ lactam (Agl) and N-aminoimidazalone (Nai) counterparts, reduced typically anti-parasitic activity; however, peptide amides possessing Agl residues at the second residue retained significant potency in the unmethylated and permethylated series. Systematic study of the effects of methylation and turn geometry on anti-parasitic activity indicated the relevance of an extended conformer about the central residues, and conformational mobility by tertiary amide isomerization and turn geometry at the extremities of the active peptides.

Leishmania infantum infection in a dog imported from Morocco.

A mixed breed dog rescued from Morocco was presented at a Quebec veterinary practice for facial lesions. Leishmaniosis, an exotic disease caused by the zoonotic protozoan Leishmania infantum, was suspected. Genomic DNA extraction from blood samples and polymerase chain reaction (PCR) were used to confirm L. infantum parasitemia. Parasites were successfully cultured from lesion biopsies, and dose-response assays demonstrated susceptibility to miltefosine, a drug that requires importation from Europe. Twenty-eight days of treatment led to the disappearance of lesions, but relapse occurred several months later (consistent with persistent parasitemia on post-treatment analysis). Further treatment would require importation of drugs and significant delays, offering a poor prognosis. Key clinical message: Diagnosis of tropical diseases in Canada will likely become more common in the near future. Having proper diagnostic tools, effective drugs, and stricter control of animal importation are essential to preventing the spread of these dangerous and frequently zoonotic diseases.

Un chien de race croisée réchappé du Maroc fut présenté dans une pratique vétérinaire du Québec pour des lésions faciales. La leishmaniose, une maladie exotique causée par le protozoaire zoonotique Leishmania infantum, fut suspectée. L'extraction d'ADN génomique d'échantillons sanguins et la réaction d'amplification en chaine par la polymérase (PCR) furent utilisées pour confirmer la parasitémie à L. infantum. Les parasites furent cultivés avec succès à partir de biopsies des lésions et des essais dose-réponse ont démontré une sensibilité au miltefosine, un médicament devant être importé d'Europe. Vingt-huit jours de traitement ont mené à la disparition des lésions, mais une rechute se produisit plusieurs mois plus tard (compatible avec une parasitémie persistante lors d'analyses post-traitement). Des traitements supplémentaires nécessiteraient l'importation de médicaments et des délais significatifs, offrant ainsi un pronostic peu optimiste.Message clinique clé :Le diagnostic de maladie tropicale au Canada devrait devenir plus fréquent dans un avenir rapproché. Il est essentiel d'avoir les outils diagnostiques appropriés, des médicaments efficaces et un contrôle plus sévère des importations d'animaux afin de prévenir la propagation de ces dangereuses et fréquentes maladies zoonotiques.(Traduit par Dr Serge Messier).

Cos-Seq for high-throughput identification of drug target and resistance mechanisms in the protozoan parasite Leishmania.

Innovative strategies are needed to accelerate the identification of antimicrobial drug targets and resistance mechanisms. Here we develop a sensitive method, which we term Cosmid Sequencing (or "Cos-Seq"), based on functional cloning coupled to next-generation sequencing. Cos-Seq identified >60 loci in the Leishmania genome that were enriched via drug selection with methotrexate and five major antileishmanials (antimony, miltefosine, paromomycin, amphotericin B, and pentamidine). Functional validation highlighted both known and previously unidentified drug targets and resistance genes, including novel roles for phosphatases in resistance to methotrexate and antimony, for ergosterol and phospholipid metabolism genes in resistance to miltefosine, and for hypothetical proteins in resistance to paromomycin, amphothericin B, and pentamidine. Several genes/loci were also found to confer resistance to two or more antileishmanials. This screening method will expedite the discovery of drug targets and resistance mechanisms and is easily adaptable to other microorganisms.

Effects of recycled manure solids bedding on the spread of gastrointestinal parasites in the environment of dairies and milk.

The primary aim of this work was to isolate common bovine digestive tract parasites in recycled manure bedding (RMS), as well as to determine the ability of current RMS preparation procedures to eliminate these pathogens. Other objectives were to assess whether any of the aforementioned parasites could be retrieved in bulk milk from dairies using RMS and to study whether the prevalence of these parasites differed among manure of cows housed on RMS versus on straw bedding. For the study, 27 RMS farms and 61 control farms were recruited. Samples of manure from the pre-pit and milk from the bulk tank were recovered from straw-bedding farms and RMS-based farms. In addition, samples from the manure solid fraction after liquid extraction, RMS before use, and RMS currently in use were recovered from RMS herds. Parasites were first detected by double centrifugation zinc sulfate flotation to enhance isolation of gastrointestinal protozoa, and by modified Wisconsin sugar flotation for the appraisal of gastrointestinal nematodes. Cryptosporidium parasites were confirmed by nested PCR amplification and sequencing of a portion of the gene encoding the small subunit rRNA. Results revealed a high prevalence of Cryptosporidium spp. (C. parvum, C. andersoni, and C. meleagridis, identified by PCR) and Eimeria spp. (mainly E. bovis and E. zuernii) parasites in both types of farms, with a larger proportion of manure samples from RMS-bedded farms testing positive for Cryptosporidium parasites compared with manure from straw-bedded farms. Both Cryptosporidium spp. and Eimeria spp. oocysts were found at every step of RMS preparation and transformation, showing that current RMS preparation strategies do not guarantee the destruction of protozoan parasites. Cryptosporidium parvum, a potential zoonotic risk for professionals in close contact with livestock, was found to be present in 32 out of 61 straw-bedded and 24 of 27 RMS farms. No protozoan parasites were found in any sample derived from bulk milk, neither by microscopy analysis nor by molecular methods.

Prevalence and geographic distribution of Echinococcus genus in wild canids in southern Québec, Canada.

Echinococcus spp. is an emerging zoonotic parasite of high concern. In Canada, an increase in the number of human and animal cases diagnosed has been reported, but information regarding the parasite's distribution in wildlife reservoir remains limited. A cross-sectional study was conducted to estimate the prevalence of wild canids infected with Echinococcus spp. and Echinococcus multilocularis in areas surrounding populated zones in Québec (Canada); to investigate the presence of areas at higher risk of infection; to evaluate potential risk factors of the infection; and as a secondary objective, to compare coproscopy and RT-PCR diagnostic tests for Taenia spp. and Echinococcus identification. From October 2020 to March 2021, fecal samples were collected from 423 coyotes (Canis latrans) and 284 red foxes (Vulpes vulpes) trapped in 12 administrative regions. Real-time PCR for molecular detection of genus Echinococcus spp. and species-specific Echinococcus multilocularis were performed. A total of 38 positive cases of Echinococcus spp., of which 25 were identified as E. multilocularis, were detected. Two high-risk areas of infection were identified. The prevalence of Echinococcus spp. was 22.7% (95% CI 11.5-37.8%) in the Montérégie centered high-risk area, 26.5% (95% CI 12.9-44.4%) in the Bas-St-Laurent high-risk area, and 3.0% (95%CI 1.8-4.7%) outside those areas. For E. multilocularis, a prevalence of 20.5% (95% CI 9.8-35.3%) was estimated in the high-risk area centered in Montérégie compared to 2.4% (95% CI 1.4-3.9%) outside. Logistic regression did not show any association of infection status with species, sex, or geolocation of capture (p > 0.05). This study shows the circulation of Echinococcus in a wildlife cycle in 9/12 administrative regions of Québec.

CRISPR-Cas9 high-throughput screening to study drug resistance in Leishmania infantum.

Parasites of the genus Leishmania pose a global health threat with limited treatment options. New drugs are urgently needed, and genomic screens have the potential to accelerate target discovery, mode of action, and resistance mechanisms against these new drugs. We describe here our effort in developing a genome-wide CRISPR-Cas9 screen in Leishmania, an organism lacking a functional nonhomologous end joining system that must rely on microhomology-mediated end joining, single-strand annealing, or homologous recombination for repairing Cas9-induced double-stranded DNA breaks. A new vector for cloning and expressing single guide RNAs (sgRNAs) was designed and proven to be effective in a small pilot project while enriching specific sgRNAs during drug selection. We then developed a whole-genome library of 49,754 sgRNAs, targeting all the genes of Leishmania infantum. This library was transfected in L. infantum expressing Cas9, and these cells were selected for resistance to two antileishmanials, miltefosine and amphotericin B. The sgRNAs the most enriched in the miltefosine screen targeted the miltefosine transporter gene, but sgRNAs targeting genes coding for a RING-variant protein and a transmembrane protein were also enriched. The sgRNAs the most enriched by amphotericin B targeted the sterol 24 C methyltransferase genes and a hypothetical gene. Through gene disruption experiments, we proved that loss of function of these genes was associated with resistance. This study describes the feasibility of carrying out whole-genome CRISPR-Cas9 screens in Leishmania provided that a strong selective pressure is applied. Such a screen can be used for accelerating the development of urgently needed antileishmanial drugs.IMPORTANCELeishmaniasis, a global health threat, lacks adequate treatment options and drug resistance exacerbates the challenge. This study introduces a CRISPR-Cas9 screening approach in Leishmania infantum, unraveling mechanisms of drug resistance at a genome-wide scale. Our screen was applied against two main antileishmanial drugs, and guides were enriched upon drug selection. These guides targeted known and new targets, hence validating the use of this screen against Leishmania. This strategy provides a powerful tool to expedite drug discovery as well as potential therapeutic targets against this neglected tropical disease.

Potential selection of antimony and methotrexate cross-resistance in Leishmania infantum circulating strains.

BACKGROUND: Visceral leishmaniasis (VL) resolution depends on a wide range of factors, including the instauration of an effective treatment coupled to a functional host immune system. Patients with a depressed immune system, like the ones receiving methotrexate (MTX), are at higher risk of developing VL and refusing antileishmanial drugs. Moreover, the alarmingly growing levels of antimicrobial resistance, especially in endemic areas, contribute to the increasing the burden of this complex zoonotic disease. PRINCIPAL FINDINGS: To understand the potential links between immunosuppressants and antileishmanial drugs, we have studied the interaction of antimony (Sb) and MTX in a Leishmania infantum reference strain (LiWT) and in two L. infantum clinical strains (LiFS-A and LiFS-B) naturally circulating in non-treated VL dogs in Spain. The LiFS-A strain was isolated before Sb treatment in a case that responded positively to the treatment, while the LiFS-B strain was recovered from a dog before Sb treatment, with the dog later relapsing after the treatment. Our results show that, exposure to Sb or MTX leads to an increase in the production of reactive oxygen species (ROS) in LiWT which correlates with a sensitive phenotype against both drugs in promastigotes and intracellular amastigotes. LiFS-A was sensitive against Sb but resistant against MTX, displaying high levels of protection against ROS when exposed to MTX. LiFS-B was resistant to both drugs. Evaluation of the melting proteomes of the two LiFS, in the presence and absence of Sb and MTX, showed a differential enrichment of direct and indirect targets for both drugs, including common and unique pathways. CONCLUSION: Our results show the potential selection of Sb-MTX cross-resistant parasites in the field, pointing to the possibility to undermine antileishmanial treatment of those patients being treated with immunosuppressant drugs in Leishmania endemic areas.

Evaluation of the Leishmania Inositol Phosphorylceramide Synthase as a Drug Target Using a Chemical and Genetic Approach.

The lack of effective vaccines and the development of resistance to the current treatments highlight the urgent need for new anti-leishmanials. Sphingolipid metabolism has been proposed as a promising source of Leishmania-specific targets as these lipids are key structural components of the eukaryotic plasma membrane and are involved in distinct cellular events. Inositol phosphorylceramide (IPC) is the primary sphingolipid in the Leishmania species and is the product of a reaction mediated by IPC synthase (IPCS). The antihistamine clemastine fumarate has been identified as an inhibitor of IPCS in L. major and a potent anti-leishmanial in vivo. Here we sought to further examine the target of this compound in the more tractable species L. mexicana, using an approach combining genomic, proteomic, metabolomic and lipidomic technologies, with molecular and biochemical studies. While the data demonstrated that the response to clemastine fumarate was largely conserved, unexpected disturbances beyond sphingolipid metabolism were identified. Furthermore, while deletion of the gene encoding LmxIPCS had little impact in vitro, it did influence clemastine fumarate efficacy and, importantly, in vivo pathogenicity. Together, these data demonstrate that clemastine does inhibit LmxIPCS and cause associated metabolic disturbances, but its primary target may lie elsewhere.

Identification and analysis of the DNA content of small extracellular vesicles isolated from Leishmania parasites.

Here, we focus on Leishmania extracellular vesicles (EVs) and their DNA content, detailing a protocol for the isolation of these nanoparticles and their subsequent genomic characterization. We describe a robust and comprehensive approach for obtaining, storing, and analyzing EVs derived from cultured parasites. We detail a user-friendly bioinformatics pipeline for sequence analysis and visualization of CNV analysis and ploidy changes. For complete details on the use and execution of this protocol, please refer to Douanne et al. (2022).1.

Pet ownership and lifestyle behaviours of immunosuppressed individuals and their relatives in the context of COVID-19 pandemic.

The COVID-19 pandemic and containment measures will likely have a detrimental impact on immunosuppressed individuals' lifestyle behaviours. Increasing evidence suggests that pet ownership is positively associated with healthier lifestyle. Yet, no study has investigated the potential benefits of pet ownership on lifestyle behaviours of immunosuppressed individuals, a population at increased risk of COVID-19 complications. This study aims to examine 1) changes in light, moderate and vigorous intensity physical activity (LPA, MPA, VPA), sedentary time (SED), and sleep duration, assessed by comparing "before COVID-19 pandemic" and "past 7 days" (i.e., current, during pandemic) self-reported behaviours in immunosuppressed individuals and their relatives; 2) to assess if changes in lifestyle behaviours are associated with pet ownership status and whether age is a moderator of these associations. A convenience sample of 132 participants (65.2% female, 41.3% ≥55 years of age) provided self-reported LPA, MPA, VPA (days/week), SED and sleep (min/day) and pet ownership status using an online questionnaire (May-August 2020). Descriptive analyses, paired T-tests, Cohen's d effect size and linear regressions were conducted. Results show that participants reported a decrease in VPA (-0.56 days/week, d = 0.34; p < 0.01) and an increase in SED (106.79 min/day, d = -0.81; p < 0.01). Stratified analysis revealed that having at least one dog, compared to not owning pets, is associated with a reduced decline in LPA, MPA and VPA and an increase in sleep in participants aged < 55 years old only. Having a dog appears to be positively associated with healthy lifestyle behaviours in younger and middle age immunosuppressed individuals.

Systematic Exploration of Functional Group Relevance for Anti-Leishmanial Activity of Anisomycin.

Assessment of structure-activity relationships for anti-protozoan activity revealed a strategy for preparing potent anisomycin derivatives with reduced host toxicity. Thirteen anisomycin analogs were synthesized by modifying the alcohol, amine, and aromatic functional groups. Examination of anti-protozoal activity against various strains of Leishmania and cytotoxicity against leucocytes with comparison against the parent natural product demonstrated typical losses of activity with modifications of the alcohol, amine, and aromatic meta-positions. On the other hand, the para-phenol moiety of anisomycin proved an effective location for introducing substituents without significant loss of anti-protozoan potency. An entry point for differentiating activity against Leishmania versus host has been uncovered by this systematic study.

Guidelines for the purification and characterization of extracellular vesicles of parasites.

Parasites are responsible for the most neglected tropical diseases, affecting over a billion people worldwide (WHO, 2015) and accounting for billions of cases a year and responsible for several millions of deaths. Research on extracellular vesicles (EVs) has increased in recent years and demonstrated that EVs shed by pathogenic parasites interact with host cells playing an important role in the parasite's survival, such as facilitation of infection, immunomodulation, parasite adaptation to the host environment and the transfer of drug resistance factors. Thus, EVs released by parasites mediate parasite-parasite and parasite-host intercellular communication. In addition, they are being explored as biomarkers of asymptomatic infections and disease prognosis after drug treatment. However, most current protocols used for the isolation, size determination, quantification and characterization of molecular cargo of EVs lack greater rigor, standardization, and adequate quality controls to certify the enrichment or purity of the ensuing bioproducts. We are now initiating major guidelines based on the evolution of collective knowledge in recent years. The main points covered in this position paper are methods for the isolation and molecular characterization of EVs obtained from parasite-infected cell cultures, experimental animals, and patients. The guideline also includes a discussion of suggested protocols and functional assays in host cells.

Sex under pressure: stress facilitates Leishmania in vitro hybridization.

The selection of Leishmania hybrids in axenic culture was considered rare until recently, when Louradour and Ferreira et al., demonstrated that induced DNA damage facilitates genetic exchange, resulting in full genome tetraploid progenies in vitro. Meiosis-related gene homologues HAP2, GEX1, and RAD51 were found to be involved, opening new avenues for functional genomic studies.

Identification of asymptomatic Leishmania infections: a scoping review.

BACKGROUND: Asymptomatic Leishmania infection may play an important role in the transmission of the parasite in endemic areas. At present there is no consensus on the definition of asymptomatic Leishmania infection, nor is there a safe and accessible gold standard test for its identification. METHODS: This paper presents a scoping review to summarize definitions of asymptomatic Leishmania infection found in the literature, as well as to detail the approach (molecular, serological, cellular, and/or parasitological tests) used by researchers to identify this asymptomatic population. A scoping review of published and gray literature related to asymptomatic Leishmania infection was conducted; retrieved citations were screened based on predefined eligibility criteria, and relevant data items were extracted from eligible articles. The analysis is descriptive and is presented using tables, figures, and thematic narrative synthesis. RESULTS: We conducted a screening of 3008 articles, of which 175 were selected for the full review. Of these articles, we selected 106 that met the inclusion criteria. These articles were published between 1991 and 2021, and in the last 5 years, up to 38 articles were reported. Most of the studies were conducted in Brazil (26%), Spain (14%), India (12%), Bangladesh (10%), and Ethiopia (7%). Of the studies, 84.9% were conducted in the immunocompetent population, while 15.1% were conducted in the immunosuppressed population (HIV, immunosuppressive drugs, and organ transplantation population). We report 14 different techniques and 10 strategies employed by researchers to define asymptomatic Leishmania infection in an endemic area. CONCLUSIONS: The definition of asymptomatic Leishmania infection is not unified across the literature, but often includes the following criteria: residence (or extended stay) in a Leishmania-endemic area, no reported signs/symptoms compatible with leishmaniasis, and positive on a combination of serological, molecular, cellular, and/or parasitological tests. Caution is recommended when comparing results of different studies on the subject of asymptomatic infections, as the reported prevalence cannot be confidently compared between areas due to the wide variety of tests employed by research groups. More research on the importance of asymptomatic immunosuppressed and immunocompetent Leishmania-positive populations in leishmaniasis epidemiology is required.

Neutrophil Extracellular Vesicles and Airway Smooth Muscle Proliferation in the Natural Model of Severe Asthma in Horses.

Extracellular vesicles (EVs) contribute to intercellular communication through the transfer of their rich cargo to recipient cells. The EVs produced by LPS-stimulated neutrophils from healthy humans and horses increase airway smooth muscle (ASM) proliferation, but the roles of neutrophil EVs in asthma are largely unexplored. The aim of this study was to determine whether neutrophil-derived EVs isolated during the remission or exacerbation of asthma influence ASM proliferation differentially. Peripheral blood neutrophils were collected during remission and exacerbation in eight horses affected by severe asthma. The cells were cultured (±LPS), and their EVs were isolated by ultracentrifugation and characterized by laser scattering microscopy and proteomic analysis. The proliferation of ASM co-incubated with EVs was monitored in real time by electrical impedance. Two proteins were significantly upregulated during disease exacerbation in neutrophil EVs (MAST4 and Lrch4), while LPS stimulation greatly altered the proteomic profile. Those changes involved the upregulation of neutrophil degranulation products, including proteases known to induce myocyte proliferation. In agreement with the proteomic results, EVs from LPS-stimulated neutrophils increased ASM proliferation, without an effect of the disease status. The inhalation of environmental LPS could contribute to asthma pathogenesis by activating neutrophils and leading to ASM hyperplasia.

Leishmania parasites exchange drug-resistance genes through extracellular vesicles.

Leishmania are eukaryotic parasites that have retained the ability to produce extracellular vesicles (EVs) through evolution. To date, it has been unclear if different DNA entities could be associated with Leishmania EVs and whether these could constitute a mechanism of horizontal gene transfer (HGT). Herein, we investigate the DNA content of EVs derived from drug-resistant parasites, as well as the EVs' potential to act as shuttles for DNA transfer. Next-generation sequencing and PCR assays confirm the enrichment of amplicons carrying drug-resistance genes associated with EVs. Transfer assays of drug-resistant EVs highlight a significant impact on the phenotype of recipient parasites induced by the expression of the transferred DNA. Recipient parasites display an enhanced growth and better control of oxidative stress. We provide evidence that eukaryotic EVs function as efficient mediators in HGT, thereby facilitating the transmission of drug-resistance genes and increasing the fitness of cells when encountering stressful environments.