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The intestinal helminth Ascaris lumbricoides infects over 800 million people. Infections are often chronic and immunity is not sterilizing due to host-immune modulation, therefore reinfection is common after antihelmintic treatment. We have previously demonstrated a role for Ascaris spp. extracellular vesicles (EVs) in host immune modulation but whether EVs are recognized by the adaptive immune system and are present systemically in the host remains unknown. Therefore, we employed a well-established trickle infection model in pigs to mimic natural Ascaris infection in humans. EVs were isolated from adult Ascaris suum followed by immunoblotting of EV and EV-depleted secretory fractions using plasma from infected and uninfected pigs. Next, EVs were isolated from pig plasma at day 56 post first infection and subjected to deep small RNAseq analysis. RNAs were aligned to A. suum and Sus scrofa miRNA complements to detect A. suum EVs and elucidate the host EV micro RNA (miRNA) response to infection, respectively. Infection generates robust antibody responses against A. suum EVs that is distinct from EV-depleted fractions. However, A. suum miRNAs were not detectable in EVs from the peripheral blood. Notably, host plasma-derived EV miRNA profiles showed significant changes between infected and uninfected pigs, indicating that Ascaris infection drives systemic changes in host EV composition.
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The Apiospora genus comprises filamentous fungi with promising potential, though its full capabilities remain undiscovered. In this study, we present the first genome assembly of an Apiospora arundinis isolate, demonstrating a highly complete and contiguous assembly estimated to 48.8 Mb, with an N99 of 3.0 Mb. Our analysis predicted a total of 15,725 genes, with functional annotations for 13,619 of them, revealing a fungus capable of producing very high amounts of carbohydrate-active enzymes (CAZymes) and secondary metabolites. Through transcriptomic analysis, we observed differential gene expression in response to varying growth media, with several genes related to carbohydrate metabolism showing significant upregulation when the fungus was cultivated on a hay-based medium. Finally, our metabolomic analysis unveiled a fungus capable of producing a diverse array of metabolites.
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Introduction: Unlike glycosylation of proteins expressed in mammalian systems, bacterial glycosylation is often neglected in the development of recombinant vaccines. Methods: Here, we compared the effects of glycosylation of YghJ, an Escherichia coli protein important for mucus attachment of bacteria causing in urinary tract infections (UTIs). A novel method based on statistical evaluation of phage display for the identification and comparison of epitopes and mimotopes of anti-YghJ antibodies in the sera was used. This is the first time that the effect of glycosylation of a recombinant bacterial antigen has been studied at the peptide epitope level. Results: The study identifies differences in the immune response for (non)-glycosylated antigens in rabbits and pigs and compares them to a large group of patients with UTI, which have been diagnosed as positive for various bacterial pathogens. We identified glycosylation-specific peptide epitopes, a large immunological similarity between different UTI pathogens, and a broad peptide epitope pattern in patients and animals, which could result in a variable response in patients upon vaccination. Discussion: This epitope analysis indicates that the vaccination of rabbits and pigs raises antibodies that translate well into the human immune system. This study underlines the importance of glycosylation in bacterial vaccines and provides detailed immune diagnostic methods to understand individual immune responses to vaccines.
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Proteínas de Escherichia coli , Infecções Urinárias , Humanos , Coelhos , Suínos , Animais , Epitopos , Antígenos de Bactérias , Glicosilação , Escherichia coli , Infecções Urinárias/microbiologia , Peptídeos , Mamíferos , MetaloproteasesRESUMO
This explorative prospective observational pilot study investigated if suggested risk factors, rectal cancer and lung metastases, could add to a relevant detection rate of asymptomatic brain metastases (BM) from colorectal cancer (CRC). Secondary, prognostic biological aspects were investigated by translational analysis of plasma samples. The study enrolled patients with rectal cancer and lung metastases. At inclusion, patients underwent a standard MRI scan of the brain. Cell-free DNA (cfDNA) level was measured by a direct fluorescence assay (DFA), and circulating tumor DNA (ctDNA) by ddPCR. BM was detected in one of twenty-nine included patients. Patients had higher cfDNA levels than healthy subjects (p < 0.01). Patients with the primary tumor in situ had higher cfDNA levels than those with resected primary tumor (p < 0.01). Patients with liver involvement had higher cfDNA levels (p = 0.12) and circulating tumor DNA levels (p = 0.01) than those without liver involvement. In conclusion, the modest incidence of BM does not justify routine MRI of the brain in this selected population. cfDNA by DFA could be a valuable tool when planning treatment and follow-up for CRC patients. Future studies should focus on identifying further characteristics and biomarkers associated with a high risk of BM, enhancing the possibility for early intervention.
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Neoplasias Encefálicas , Ácidos Nucleicos Livres , DNA Tumoral Circulante , Neoplasias Colorretais , Neoplasias Pulmonares , Neoplasias Retais , Humanos , Estudos Prospectivos , Neoplasias Pulmonares/genética , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/genética , Biomarcadores Tumorais/genética , Neoplasias Colorretais/patologiaRESUMO
BACKGROUND: Patients with detectable ctDNA after radical-intent treatment of metastatic spread from colorectal cancer (mCRC) have a very high risk of recurrence, which may be prevented with intensified adjuvant chemotherapy (aCTh). In the OPTIMISE study, we investigate ctDNA-guided aCTh after radical-intent treatment of mCRC. Here we present results from the preplanned interim analysis. MATERIAL AND METHODS: The study is an open-label 1:1 randomized clinical trial comparing ctDNA-guided aCTh against standard of care (SOC), with a run-in phase investigating feasibility measures. Key inclusion criteria; radical-intent treatment for mCRC and clinically eligible for triple-agent chemotherapy. Patients underwent a PET-CT scan before randomization. ctDNA analyses of plasma samples were done by ddPCR, detecting CRC-specific mutations and methylation of the NPY gene. In the ctDNA-guided arm, ctDNA positivity led to an escalation strategy with triple-agent chemotherapy, and conversely ctDNA negativity led to a de-escalation strategy by shared-decision making. Patients randomized to the standard arm were treated according to SOC. Feasibility measures for the run-in phase were; the inclusion of 30 patients over 12 months in two Danish hospitals, compliance with randomization >80%, rate of PET-CT-positive findings <20%, and eligibility for triple-agent chemotherapy >80%. RESULTS: Thirty-two patients were included. The rate of PET-CT-positive cases was 22% (n = 7/32). Ninety-seven percent of the patients were randomized. Fourteen patients were randomly assigned to SOC and sixteen to ctDNA-guided adjuvant treatment and follow-up. All analyses of baseline plasma samples in the ctDNA-guided arm passed the quality control, and 19% were ctDNA positive. The median time to result was three working days. All ctDNA-positive patients were eligible for triple-agent chemotherapy. CONCLUSION: The study was proven to be feasible and continues in the planned large-scale phase II trial. Results from the OPTIMISE study will potentially optimize the adjuvant treatment of patients undergoing radical-intent treatment of mCRC, thereby improving survival and reducing chemotherapy-related toxicity.
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Neoplasias Colorretais , Humanos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Biomarcadores Tumorais/genética , Hormônio AdrenocorticotrópicoRESUMO
INTRODUCTION: Both quantitative and molecular changes in ctDNA can hold important information when treating metastatic colorectal cancer (mCRC), but its clinical utility is yet to be established. Before conducting a large-scale randomized trial, it is essential to test feasibility. This study investigates whether ctDNA is feasible for detecting patients who will benefit from treatment with epidermal growth factor receptor inhibitors and the prognostic value of circulating tumor DNA (ctDNA) response. MATERIALS AND METHODS: Patients with mCRC, who were considered for systemic palliative treatment and were eligible for ctDNA analysis. Mutational testing on cell-free DNA (cfDNA) was done by ddPCR. ctDNA response from baseline to the third treatment cycle was evaluated in patients with detectable ctDNA at baseline. ctDNA maximum response was defined as undetectable ctDNA at the third treatment cycle, ctDNA partial response as any decrease in the ctDNA level, and ctDNA progression as any increase in the ctDNA level. RESULTS: Forty-nine patients were included. The time to test results for mutational testing on cfDNA was significantly shorter than on tumor tissue (p < .001). Progression-free survival were 11.2 months (reference group), 7.5 months (HR = 10.7, p= .02), and 4.6 months (HR = 11.4, p= .02) in patients with ctDNA maximum response, partial response, and progression, respectively. Overall survival was 31.2 months (reference group), 15.2 months (HR = 4.1, p= .03), and 9.0 months (HR = 2.6, p= .03) in patients with ctDNA maximum response, partial response, and progression, respectively. CONCLUSION: Pretreatment mutational testing on cfDNA in daily clinic is feasible and can be applied in randomized clinical trials evaluating the clinical utility of ctDNA. Early dynamics in ctDNA during systemic treatment hold prognostic value.
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DNA Tumoral Circulante , Neoplasias Colorretais , Humanos , Estudos de Viabilidade , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Prognóstico , DNA Tumoral Circulante/genética , Mutação , Biomarcadores Tumorais/genéticaRESUMO
Polymyxin B (PMB) is a peptide based antibiotic that binds the lipid A moiety of lipopolysaccharide (LPS) with a resultant bactericidal effect. The interaction of PMB with LPS presented on outer membrane vesicles (OMVs) is not fully known, however, a sacrificial role of OMVs in protecting bacterial cells by sequestering PMB has been described. Here we assess the ability of PMB to neutralize the immune-stimulatory properties of OMVs whilst modulating the uptake of OMVs in human immune cells. We show for the first time that PMB increases immune cell uptake of Escherichia coli derived OMVs whilst inhibiting TNF and IL-1ß production. Therefore, we present a potential new role for PMB in the neutralization of OMVs via LPS masking and increased immune cell uptake.
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Escherichia coli , Polimixina B , Humanos , Polimixina B/farmacologia , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Antibacterianos/farmacologia , Peptídeos/farmacologiaRESUMO
Over the last decade, research interest in defining how extracellular vesicles (EVs) shape cross-species communication has grown rapidly. Parasitic helminths, worm species found in the phyla Nematoda and Platyhelminthes, are well-recognised manipulators of host immune function and physiology. Emerging evidence supports a role for helminth-derived EVs in these processes and highlights EVs as an important participant in cross-phylum communication. While the mammalian EV field is guided by a community-agreed framework for studying EVs derived from model organisms or cell systems [e.g., Minimal Information for Studies of Extracellular Vesicles (MISEV)], the helminth community requires a supplementary set of principles due to the additional challenges that accompany working with such divergent organisms. These challenges include, but are not limited to, generating sufficient quantities of EVs for descriptive or functional studies, defining pan-helminth EV markers, genetically modifying these organisms, and identifying rigorous methodologies for in vitro and in vivo studies. Here, we outline best practices for those investigating the biology of helminth-derived EVs to complement the MISEV guidelines. We summarise community-agreed standards for studying EVs derived from this broad set of non-model organisms, raise awareness of issues associated with helminth EVs and provide future perspectives for how progress in the field will be achieved.
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Vesículas Extracelulares , Helmintos , Animais , Humanos , Vesículas Extracelulares/fisiologia , Reprodutibilidade dos Testes , MamíferosRESUMO
BACKGROUND: We investigate the current knowledge on circulating tumour DNA (ctDNA) and its clinical utility in predicting outcomes in patients with metastatic colorectal cancer (mCRC). METHODS: PubMed, Embase, Cochrane Database of Systematic Reviews and Cochrane Central Register of Controlled Trials were searched. Last search 16/12/2020. We included studies on patients with mCRC reporting the predictive or prognostic value of ctDNA. We performed separate random-effects meta-analyses to investigate if baseline ctDNA and early changes in ctDNA levels during treatment were associated with survival. The risk of bias was assessed according to the Quality in Prognosis Studies tool. RESULTS: Seventy-one studies were included with 6930 patients. Twenty-four studies were included in meta-analyses. High baseline ctDNA level was associated with short progression-free survival (PFS) (HR = 2.2; 95% CI 1.8-2.8; n = 509) and overall survival (OS) (HR = 2.4; 95% CI 1.9-3.1; n = 1336). A small or no early decrease in ctDNA levels during treatment was associated with short PFS (HR = 3.0; 95% CI 2.2-4.2; n = 479) and OS (HR = 2.8; 95% CI 2.1-3.9; n = 583). Results on clonal evolution and lead-time were inconsistent. A majority of included studies (n = 50/71) had high risk of bias in at least one domain. CONCLUSIONS: Plasma ctDNA is a strong prognostic biomarker in mCRC. However, true clinical utility is lacking.
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DNA Tumoral Circulante , Neoplasias Colorretais , DNA Tumoral Circulante/genética , Neoplasias Colorretais/patologia , Humanos , Prognóstico , Intervalo Livre de ProgressãoRESUMO
Treatment for metastatic colorectal cancer (mCRC) is focused on prolonging survival and maintaining quality of life. It is important to establish prognostic and predictive markers to avoid extended, ineffective treatment. The aim of the present study was, by a novel approach, to analyze the association between cell-free (cf)DNA levels and outcome in patients receiving systemic therapy for incurable mCRC. The study was a prospective non-interventional biomarker study for patients receiving standard of systemic treatment for mCRC. Patients with mCRC, who, according to standard guidelines, were considered for treatment with EGFR inhibitors, were included. The cfDNA levels in consecutive plasma samples were measured by a direct fluorescence assay. The study included 47 patients. Blood samples were available at baseline (n=47); prior to the third treatment cycle (n=31); the first (n=33), second (n=22) and third response evaluation during treatment (n=17); and at progression (n=22). The disease control rate was 42 and 91% in patients with high (≥75th percentile of baseline cfDNA levels) and low cfDNA levels (<75th percentile of baseline cfDNA levels), respectively (P<0.001). Median progression-free survival (PFS) was 3.8 and 8.5 months in patients with high and low cfDNA levels, respectively (hazard ratio=3.03, 95% CI 1.46-6.29, P<0.01). Median overall survival (OS) was 5.0 and 26.6 months in patients with high and low cfDNA levels, respectively (hazard ratio=3.48, 95% CI 1.44-8.44, P<0.01). In the multivariate analysis, baseline cfDNA levels remained a significant predictor of PFS and OS. In conclusion, cfDNA is a promising prognostic tool in the personalized treatment of mCRC. cfDNA levels were estimated by a simple, rapid and inexpensive method (OPTIPAL II: ClinicalTrials.gov identifier no. NCT03750175; registered November 21, 2018).
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Extracellular vesicles (EVs) large-scale production is a crucial point for the translation of EVs from discovery to application of EV-based products. In October 2021, the International Society for Extracellular Vesicles (ISEV), along with support by the FET-OPEN projects, "The Extracellular Vesicle Foundry" (evFOUNDRY) and "Extracellular vesicles from a natural source for tailor-made nanomaterials" (VES4US), organized a workshop entitled "massivEVs" to discuss the potential challenges for translation of EV-based products. This report gives an overview of the topics discussed during "massivEVs", the most important points raised, and the points of consensus reached after discussion among academia and industry representatives. Overall, the review of the existing EV manufacturing, upscaling challenges and directions for their resolution highlighted in the workshop painted an optimistic future for the expanding EV field.
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Helminths survive within their host by secreting immunomodulatory compounds, which hold therapeutic potential for inflammatory conditions. Helminth-derived extracellular vesicles (EVs) are one such component proposed to possess immunomodulatory activities. Due to the recent discovery of helminth EVs, standardised protocols for EV separation are lacking. Excretory/secretory products of the porcine helminth, Ascaris suum, were used to compare three EV separation methods: Size exclusion chromatography (SEC), ultracentrifugation (UC) and a combination of the two. Their performance was evaluated by EV yield, sample purity and the ability of EVs to suppress lipopolysaccharide (LPS)-induced inflammation in vitro. We found that all three separation methods successfully separated helminth EVs with a similar EV yield. Functional studies showed that EVs from all three methods reduced LPS-induced levels of tumour necrosis factor (TNF-α) in a dose-dependent manner. Overall, the three separation methods showed similar performance, however, the combination of UC+SEC presented with slightly higher purity than either method alone.
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Enterotoxigenic Escherichia coli (ETEC) is a WHO priority pathogen and vaccine target which causes infections in low-income and middle-income countries, travelers visiting endemic regions. The global urgent demand for an effective preventive intervention has become more pressing as ETEC strains have become increasingly multiple antibiotic resistant. However, the vaccine development pipeline has been slow to address this urgent need. To date, vaccine development has focused mainly on canonical antigens such as colonization factors and expressed toxins but due to genomic plasticity of this enteric pathogen, it has proven difficult to develop effective vaccines. In this study, we investigated the highly conserved non-canonical vaccine candidate YghJ/SsLE. Using the mass spectrometry-based method BEMAP, we demonstrate that YghJ is hyperglycosylated in ETEC and identify 54 O-linked Set/Thr residues within the 1519 amino acid primary sequence. The glycosylation sites are evenly distributed throughout the sequence and do not appear to affect the folding of the overall protein structure. Although the glycosylation sites only constitute a minor subpopulation of the available epitopes, we observed a notable difference in the immunogenicity of the glycosylated YghJ and the non-glycosylated protein variant. We can demonstrate by ELISA that serum from patients enrolled in an ETEC H10407 controlled infection study are significantly more reactive with glycosylated YghJ compared to the non-glycosylated variant. This study provides an important link between O-linked glycosylation and the relative immunogenicity of bacterial proteins and further highlights the importance of this observation in considering ETEC proteins for inclusion in future broad coverage subunit vaccine candidates.
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Escherichia coli Enterotoxigênica , Infecções por Escherichia coli , Proteínas de Escherichia coli , Metaloproteases , Antígenos de Bactérias , Epitopos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glicosilação , Humanos , Metaloproteases/genética , Metaloproteases/metabolismoRESUMO
Efforts to develop broadly protective vaccines against pathogenic Escherichia coli are ongoing. A potential antigen candidate for vaccine development is the metalloprotease YghJ, or SslE. YghJ is a conserved mucinase that is immunogenic, heavily glycosylated, and produced by most pathogenic E. coli. To develop efficacious YghJ-based vaccines, there is a need to investigate to what extent potentially protective antibody responses target glycosylated epitopes in YghJ and to describe variations in the quality of YghJ glycosylation in the E. coli population. In this study we estimated the proportion of anti-YghJ IgA antibodies that targeted glycosylated epitopes in serum and intestinal lavage samples from 21 volunteers experimentally infected with wild-type enterotoxigenic E. coli (ETEC) strain TW10722. Glycosylated and non-glycosylated YghJ was expressed, purified, and then gycosylation pattern was verified by BEMAP analysis. Then we used a multiplex bead flow cytometric assay to analyse samples from before and 10 days after TW10722 was ingested. We found that 20 (95%) of the 21 volunteers had IgA antibody responses to homologous, glycosylated YghJ, with a median fold increase in IgA levels of 7.9 (interquartile range [IQR]: 7.1, 11.1) in serum and 3.7 (IQR: 2.1, 10.7) in lavage. The median proportion of anti-YghJ IgA response that specifically targeted glycosylated epitopes was 0.45 (IQR: 0.30, 0.59) in serum and 0.07 (IQR: 0.01, 0.22) in lavage. Our findings suggest that a substantial, but variable, proportion of the IgA antibody response to YghJ in serum during ETEC infection is targeted against glycosylated epitopes, but that gut IgA responses largely target non-glycosylated epitopes. Further research into IgA targeting glycosylated YghJ epitopes is of interest to the vaccine development efforts.
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Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/imunologia , Infecções por Escherichia coli/imunologia , Proteínas de Escherichia coli/imunologia , Imunoglobulina A/imunologia , Metaloproteases/imunologia , Anticorpos Antibacterianos/sangue , Escherichia coli Enterotoxigênica , Epitopos/imunologia , Infecções por Escherichia coli/sangue , Proteínas de Escherichia coli/genética , Glicosilação , Humanos , Imunidade nas Mucosas , Imunoglobulina A/sangue , Intestinos/imunologia , Metaloproteases/genéticaRESUMO
Hepatic arterial infusion (HAI) of chemotherapy is an experimental treatment option for patients with colorectal cancer liver metastases (CRCLM). The current study aimed to investigate the predictive and prognostic value of cell free DNA (cfDNA) in patients with CRCLM receiving HAI with oxaliplatin and systemic capecitabine. Plasma samples from 62 patients were investigated who were included into a single arm phase II study investigating HAI treatment for patients with CRCLM. The clinical outcome of the trial has been presented previously. In brief, treatment consisted of intrahepatic infusion of oxaliplatin 100 mg/m2 every second week with concomitant oral capecitabine 3,500 mg/m2 every second week for up to 12 cycles. Blood samples were drawn at baseline and follow-up and plasma was analyzed for cell free DNA using a direct fluorescent assay. The baseline level of plasma cfDNA was 0.92 ng/µl (95% CI 0.84-1.00). Patients with a baseline value of cfDNA above the 75th quartile had a median overall survival of 2.4 years (95% CI 0.7-2.8), compared with 3.9 years (95% CI 2.8-5.9) for patients below the 75th quartile (P=0.02). The baseline level of cfDNA was significantly lower (0.91 ng/µl, 95% CI 0.76-0.98) in patients who achieved an objective response compared to non-responders (1.79 ng/µl; 95% CI 0.99-2.57; P=0.02). The current study demonstrated a possible prognostic and predictive value of cfDNA for patients with CRCLM undergoing HAI with oxaliplatin and concomitant capecitabine.
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BACKGROUND: Local treatment of liver and/or lung metastases from colorectal cancer (CRC) is increasingly used in daily practice and comprises resection, radiofrequency ablation (RFA) and stereotactic radiotherapy (SBRT). The need for prognostic markers for patients undergoing such treatment is currently unmet. We investigated post-treatment circulating tumor-specific DNA (ctDNA) analysis and address a possible prognostic value in a pilot study. MATERIALS: From July 2015 to September 2017, patients undergoing standard of care local treatment of liver and/or lung metastases were included in a prospective translational study. Blood samples were drawn 2 weeks after local treatment and during follow-up. CtDNA was detected by ddPCR and a mass spectrometry-based platform MassARRAY®. RESULTS: Post treatment blood samples were available for 35 patients including five with detectable ctDNA (KRAS mutation, n = 2; NRAS mutation, n = 2; BRAF mutation, n = 1) by ddPCR. 17 out of 35 patients (49%) developed recurrence within a median of 273 days (95%CI 95-NA) among patients positive for ctDNA, while the median time to recurrence was not reached for the group of patients negative for ctDNA (p = .03). CONCLUSION: The presence of ctDNA following local treatment of metastatic CRC is associated with an increased risk of recurrence and a short time to failure.
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DNA Tumoral Circulante , Neoplasias do Colo , Neoplasias Colorretais , Biomarcadores Tumorais/genética , DNA Tumoral Circulante/genética , Neoplasias Colorretais/genética , Humanos , Recidiva Local de Neoplasia/genética , Neoplasia Residual , Projetos Piloto , Estudos ProspectivosRESUMO
In the last two decades, extracellular vesicles (EVs) from the three domains of life, Archaea, Bacteria and Eukaryotes, have gained increasing scientific attention. As such, the role of EVs in host-pathogen communication and immune modulation are being intensely investigated. Pivotal to EV research is the determination of how and where EVs are taken up by recipient cells and organs in vivo, which requires suitable tracking strategies including labelling. Labelling of EVs is often performed post-isolation which increases risks of non-specific labelling and the introduction of labelling artefacts. Here we exploited the inability of helminths to de novo synthesise fatty acids to enable labelling of EVs by whole organism uptake of fluorescent lipid analogues and the subsequent incorporation in EVs. We showed uptake of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (DOPE-Rho) in Anisakis spp. and Trichuris suis larvae. EVs isolated from the supernatant of Anisakis spp. labelled with DOPE-Rho were characterised to assess the effects of labelling on size, structure and fluorescence of EVs. Fluorescent EVs were successfully taken up by the human macrophage cell line THP-1. This study, therefore, presents a novel staining method that can be utilized by the EV field in parasitology and potentially across multiple species.
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The mechanical properties of extracellular vesicles (EVs) are known to influence their biological function, in terms of, e.g., cellular adhesion, endo/exocytosis, cellular uptake, and mechanosensing. EVs have a characteristic nanomechanical response which can be probed via force spectroscopy (FS) and exploited to single them out from nonvesicular contaminants or to discriminate between subtypes. However, measuring the nanomechanical characteristics of individual EVs via FS is a labor-intensive and time-consuming task, usually limiting this approach to specialists. Herein, we describe a simple atomic force microscopy based experimental procedure for the simultaneous nanomechanical and morphological analysis of several hundred individual nanosized EVs within the hour time scale, using basic AFM equipment and skills and only needing freely available software for data analysis. This procedure yields a "nanomechanical snapshot" of an EV sample which can be used to discriminate between subpopulations of vesicular and nonvesicular objects in the same sample and between populations of vesicles with similar sizes but different mechanical characteristics. We demonstrate the applicability of the proposed approach to EVs obtained from three very different sources (human colorectal carcinoma cell culture, raw bovine milk, and Ascaris suum nematode excretions), recovering size and stiffness distributions of individual vesicles in a sample. EV stiffness values measured with our high-throughput method are in very good quantitative accord with values obtained by FS techniques which measure EVs one at a time. We show how our procedure can detect EV samples contamination by nonvesicular aggregates and how it can quickly attest the presence of EVs even in samples for which no established assays and/or commercial kits are available (e.g., Ascaris EVs), thus making it a valuable tool for the rapid assessment of EV samples during the development of isolation/enrichment protocols by EV researchers. As a side observation, we show that all measured EVs have a strikingly similar stiffness, further reinforcing the hypothesis that their mechanical characteristics could have a functional role.
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Vesículas Extracelulares/química , Ensaios de Triagem em Larga Escala , Microscopia de Força Atômica , Nanotecnologia , Animais , Ascaris suum/química , Bovinos , Células HCT116 , Humanos , Lipossomos/química , Leite/químicaRESUMO
Although the study of helminth-derived extracellular vesicles (EVs) is in its infancy, proteomic studies of EVs from representatives of nematodes, cestodes and trematodes have identified homologs of mammalian EV proteins including components of the endosomal sorting complexes required for transport and heat-shock proteins, suggesting conservation of pathways of EV biogenesis and cargo loading between helminths and their hosts. However, parasitic helminth biology is unique and this is likely reflected in helminth EV composition and biological activity. This opinion article highlights two exceptional studies that identified EVs released by Heligmosomoides polygyrus and Fasciola hepatica which display differential lipid and glycan composition, respectively, when compared with EVs derived from mammalian cells. Furthermore, we discuss the potential implications of helminth EV lipid and glycan composition upon helminth infection and host pathology. Future studies, focusing on the unique composition and functional properties of helminth EVs, may prove crucial to the understanding of host-parasite communication.