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Background: Ascaris lumbricoides cystatin (Al-CPI) prevents the development of allergic airway inflammation and dextran-induced colitis in mice models. It has been suggested that helminth-derived cystatins inhibit cathepsins in dendritic cells (DC), but their immunomodulatory mechanisms are unclear. We aimed to analyze the transcriptional profile of human monocyte-derived DC (moDC) upon stimulation with Al-CPI to elucidate target genes and pathways of parasite immunomodulation. Methods: moDC were generated from peripheral blood monocytes from six healthy human donors of Denmark, stimulated with 1 µM of Al-CPI, and cultured for 5 hours at 37°C. RNA was sequenced using TrueSeq RNA libraries and the NextSeq 550 v2.5 (75 cycles) sequencing kit (Illumina, Inc). After QC, reads were aligned to the human GRCh38 genome using Spliced Transcripts Alignment to a Reference (STAR) software. Differential expression was calculated by DESEq2 and expressed in fold changes (FC). Cell surface markers and cytokine production by moDC were evaluated by flow cytometry. Results: Compared to unstimulated cells, Al-CPI stimulated moDC showed differential expression of 444 transcripts (|FC| ≥1.3). The top significant differences were in Kruppel-like factor 10 (KLF10, FC 3.3, PBH = 3 x 10-136), palladin (FC 2, PBH = 3 x 10-41), and the low-density lipoprotein receptor (LDLR, FC 2.6, PBH = 5 x 10-41). Upregulated genes were enriched in regulation of cholesterol biosynthesis by sterol regulatory element-binding proteins (SREBP) signaling pathways and immune pathways. Several genes in the cholesterol biosynthetic pathway showed significantly increased expression upon Al-CPI stimulation, even in the presence of lipopolysaccharide (LPS). Regarding the pathway of negative regulation of immune response, we found a significant decrease in the cell surface expression of CD86, HLA-DR, and PD-L1 upon stimulation with 1 µM Al-CPI. Conclu

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., 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.

Background Over a billion people are infected with Toxocara canis or T. cati, the roundworms of dogs and cats. Historically,T. canis has been considered the main species responsible for human toxocarosis, but as serodiagnosis cannot discriminatebetween the two species, this remains unresolved. We used pigs as a relevant large animal model for human infection toassess the migratory pattern of T. cati and T. canis.Methods Pigs were inoculated with T. cati or T. canis eggs or PBS (negative controls) and necropsied 14 or 31 days later.Different organs and tissues were examined for parasites and pathological changes.Results Overall, the two parasite species had a similar migration pattern reaching multiple organs and tissues, including themesenteric lymph nodes, liver, lungs, and diaphragm. We recovered larvae of both species in the brain, suggesting that T.cati also can cause neurological toxocarosis in humans. Both species induced systemic eosinophilia and histopathologicalchanges in the lungs, livers, and mesenteric lymph nodes.Conclusion This study emphasises the importance of T. cati as a zoonotic agent and the need to develop diagnostic methodsthat can differentiate between sources of infection in humans., Background: Over a billion people are infected with Toxocara canis or T. cati, the roundworms of dogs and cats. Historically, T. canis has been considered the main species responsible for human toxocarosis, but as serodiagnosis cannot discriminate between the two species, this remains unresolved. We used pigs as a relevant large animal model for human infection to assess the migratory pattern of T. cati and T. canis. Methods: Pigs were inoculated with T. cati or T. canis eggs or PBS (negative controls) and necropsied 14 or 31 days later. Different organs and tissues were examined for parasites and pathological changes. Results: Overall, the two parasite species had a similar migration pattern reaching multiple organs and tissues, including the mesenteric lymph nodes, liver, lungs, and diaphragm. We recovered larvae of both species in the brain, suggesting that T. cati also can cause neurological toxocarosis in humans. Both species induced systemic eosinophilia and histopathological changes in the lungs, livers, and mesenteric lymph nodes. Conclusion: This study emphasises the importance of T. cati as a zoonotic agent and the need to develop diagnostic methods that can differentiate between sources of infection in humans.

Background: Ascaris lumbricoides cystatin (Al-CPI) prevents the development of allergic airway inflammation and dextran-induced colitis in mice models. It has been suggested that helminth-derived cystatins inhibit cathepsins in dendritic cells (DC), but their immunomodulatory mechanisms are unclear. We aimed to analyze the transcriptional profile of human monocyte-derived DC (moDC) upon stimulation with Al-CPI to elucidate target genes and pathways of parasite immunomodulation. Methods: moDC were generated from peripheral blood monocytes from six healthy human donors of Denmark, stimulated with 1 µM of Al-CPI, and cultured for 5 hours at 37°C. RNA was sequenced using TrueSeq RNA libraries and the NextSeq 550 v2.5 (75 cycles) sequencing kit (Illumina, Inc). After QC, reads were aligned to the human GRCh38 genome using Spliced Transcripts Alignment to a Reference (STAR) software. Differential expression was calculated by DESEq2 and expressed in fold changes (FC). Cell surface markers and cytokine production by moDC were evaluated by flow cytometry. Results: Compared to unstimulated cells, Al-CPI stimulated moDC showed differential expression of 444 transcripts (|FC| ≥1.3). The top significant differences were in Kruppel-like factor 10 (KLF10, FC 3.3, PBH = 3 x 10-136), palladin (FC 2, PBH = 3 x 10-41), and the low-density lipoprotein receptor (LDLR, FC 2.6, PBH = 5 x 10-41). Upregulated genes were enriched in regulation of cholesterol biosynthesis by sterol regulatory element-binding proteins (SREBP) signaling pathways and immune pathways. Several genes in the cholesterol biosynthetic pathway showed significantly increased expression upon Al-CPI stimulation, even in the presence of lipopolysaccharide (LPS). Regarding the pathway of negative regulation of immune response, we found a significant decrease in the cell surface expression of CD86, HLA-DR, and PD-L1 upon stimulation with 1 µM Al-CPI. Conclu

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., 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.

Background Over a billion people are infected with Toxocara canis or T. cati, the roundworms of dogs and cats. Historically,T. canis has been considered the main species responsible for human toxocarosis, but as serodiagnosis cannot discriminatebetween the two species, this remains unresolved. We used pigs as a relevant large animal model for human infection toassess the migratory pattern of T. cati and T. canis.Methods Pigs were inoculated with T. cati or T. canis eggs or PBS (negative controls) and necropsied 14 or 31 days later.Different organs and tissues were examined for parasites and pathological changes.Results Overall, the two parasite species had a similar migration pattern reaching multiple organs and tissues, including themesenteric lymph nodes, liver, lungs, and diaphragm. We recovered larvae of both species in the brain, suggesting that T.cati also can cause neurological toxocarosis in humans. Both species induced systemic eosinophilia and histopathologicalchanges in the lungs, livers, and mesenteric lymph nodes.Conclusion This study emphasises the importance of T. cati as a zoonotic agent and the need to develop diagnostic methodsthat can differentiate between sources of infection in humans., Background: Over a billion people are infected with Toxocara canis or T. cati, the roundworms of dogs and cats. Historically, T. canis has been considered the main species responsible for human toxocarosis, but as serodiagnosis cannot discriminate between the two species, this remains unresolved. We used pigs as a relevant large animal model for human infection to assess the migratory pattern of T. cati and T. canis. Methods: Pigs were inoculated with T. cati or T. canis eggs or PBS (negative controls) and necropsied 14 or 31 days later. Different organs and tissues were examined for parasites and pathological changes. Results: Overall, the two parasite species had a similar migration pattern reaching multiple organs and tissues, including the mesenteric lymph nodes, liver, lungs, and diaphragm. We recovered larvae of both species in the brain, suggesting that T. cati also can cause neurological toxocarosis in humans. Both species induced systemic eosinophilia and histopathological changes in the lungs, livers, and mesenteric lymph nodes. Conclusion: This study emphasises the importance of T. cati as a zoonotic agent and the need to develop diagnostic methods that can differentiate between sources of infection in humans.

The neglected tropical disease trichuriasis is caused by the whipworm Trichuris trichiura, a soil-transmitted helminth that has infected humans for millennia. Today, T. trichiura infects as many as 500 million people, predominantly in communities with poor sanitary infrastructure enabling sustained faecal-oral transmission. Using whole-genome sequencing of geographically distributed worms collected from human and other primate hosts, together with ancient samples preserved in archaeologically-defined latrines and deposits dated up to one thousand years old, we present the first population genomics study of T. trichiura. We describe the continent-scale genetic structure between whipworms infecting humans and baboons relative to those infecting other primates. Admixture and population demographic analyses support a stepwise distribution of genetic variation that is highest in Uganda, consistent with an African origin and subsequent translocation with human migration. Finally, genome-wide analyses between human samples and between human and non-human primate samples reveal local regions of genetic differentiation between geographically distinct populations. These data provide insight into zoonotic reservoirs of human-infective T. trichiura and will support future efforts toward the implementation of genomic epidemiology of this globally important helminth.

The neglected tropical disease trichuriasis is caused by the whipworm Trichuris trichiura, a soil-transmitted helminth that has infected humans for millennia. Today, T. trichiura infects as many as 500 million people, predominantly in communities with poor sanitary infrastructure enabling sustained faecal-oral transmission. Using whole-genome sequencing of geographically distributed worms collected from human and other primate hosts, together with ancient samples preserved in archaeologically-defined latrines and deposits dated up to one thousand years old, we present the first population genomics study of T. trichiura. We describe the continent-scale genetic structure between whipworms infecting humans and baboons relative to those infecting other primates. Admixture and population demographic analyses support a stepwise distribution of genetic variation that is highest in Uganda, consistent with an African origin and subsequent translocation with human migration. Finally, genome-wide analyses between human samples and between human and non-human primate samples reveal local regions of genetic differentiation between geographically distinct populations. These data provide insight into zoonotic reservoirs of human-infective T. trichiura and will support future efforts toward the implementation of genomic epidemiology of this globally important helminth.

BACKGROUND: Infections with Ascaris lumbricoides and Trichuris trichiura remain significant contributors to the global burden of neglected tropical diseases. Infection may in particular affect child development as they are more likely to be infected with T. trichiura and/or A. lumbricoides and to carry higher worm burdens than adults. Whilst the impact of heavy infections are clear, the effects of moderate infection intensities on the growth and development of children remain elusive. Field studies are confounded by a lack of knowledge of infection history, nutritional status, presence of co-infections and levels of exposure to infective eggs. Therefore, animal models are required. Given the physiological similarities between humans and pigs but also between the helminths that infect them; A. suum and T. suis, growing pigs provide an excellent model to investigate the direct effects of Ascaris spp. and Trichuris spp. on weight gain. METHODS AND RESULTS: We employed a trickle infection protocol to mimic natural co-infection to assess the effect of infection intensity, determined by worm count (A. suum) or eggs per gram of faeces (A. suum and T. suis), on weight gain in a large pig population (n = 195) with variable genetic susceptibility. Pig body weights were assessed over 14 weeks. Using a post-hoc statistical approach, we found a negative association between weight gain and T. suis infection. For A. suum, this association was not significant after adjusting for other covariates in a multivariable analysis. Estimates from generalized linear mixed effects models indicated that a 1 kg increase in weight gain was associated with 4.4% (p = 0.00217) decrease in T. suis EPG and a 2.8% (p = 0.02297) or 2.2% (p = 0.0488) decrease in A. suum EPG or burden, respectively. CONCLUSIONS: Overall this study has demonstrated a negative association between STH and weight gain in growing pigs but also that T. suis infection may be more detrimental that A. suum on growth.

BACKGROUND: Infections with Ascaris lumbricoides and Trichuris trichiura remain significant contributors to the global burden of neglected tropical diseases. Infection may in particular affect child development as they are more likely to be infected with T. trichiura and/or A. lumbricoides and to carry higher worm burdens than adults. Whilst the impact of heavy infections are clear, the effects of moderate infection intensities on the growth and development of children remain elusive. Field studies are confounded by a lack of knowledge of infection history, nutritional status, presence of co-infections and levels of exposure to infective eggs. Therefore, animal models are required. Given the physiological similarities between humans and pigs but also between the helminths that infect them; A. suum and T. suis, growing pigs provide an excellent model to investigate the direct effects of Ascaris spp. and Trichuris spp. on weight gain. METHODS AND RESULTS: We employed a trickle infection protocol to mimic natural co-infection to assess the effect of infection intensity, determined by worm count (A. suum) or eggs per gram of faeces (A. suum and T. suis), on weight gain in a large pig population (n = 195) with variable genetic susceptibility. Pig body weights were assessed over 14 weeks. Using a post-hoc statistical approach, we found a negative association between weight gain and T. suis infection. For A. suum, this association was not significant after adjusting for other covariates in a multivariable analysis. Estimates from generalized linear mixed effects models indicated that a 1 kg increase in weight gain was associated with 4.4% (p = 0.00217) decrease in T. suis EPG and a 2.8% (p = 0.02297) or 2.2% (p = 0.0488) decrease in A. suum EPG or burden, respectively. CONCLUSIONS: Overall this study has demonstrated a negative association between STH and weight gain in growing pigs but also that T. suis infection may be more detrimental that A. suum on growth.

The neglected tropical disease trichuriasis is caused by the whipworm Trichuris trichiura, a soil-transmitted helminth that has infected humans for millennia. Today, T. trichiura infects as many as 500 million people, predominantly in communities with poor sanitary infrastructure enabling sustained faecal-oral transmission. Using whole-genome sequencing of geographically distributed worms collected from human and other primate hosts, together with ancient samples preserved in archaeologically-defined latrines and deposits dated up to one thousand years old, we present the first population genomics study of T. trichiura. We describe the continent-scale genetic structure between whipworms infecting humans and baboons relative to those infecting other primates. Admixture and population demographic analyses support a stepwise distribution of genetic variation that is highest in Uganda, consistent with an African origin and subsequent translocation with human migration. Finally, genome-wide analyses between human samples and between human and non-human primate samples reveal local regions of genetic differentiation between geographically distinct populations. These data provide insight into zoonotic reservoirs of human-infective T. trichiura and will support future efforts toward the implementation of genomic epidemiology of this globally important helminth.

Regulation of macrophage (Mɸ) function can maintain tissue homeostasis and control inflammation. Parasitic worms (helminths) are potent modulators of host immune and inflammatory responses. They have evolved various strategies to promote immunosuppression, including redirecting phagocytic cells toward a regulatory phenotype. Although soluble products from the whipworm Trichuris suis (TSPs) have shown significant effects on Mɸ function, the mechanisms underlying these modulatory effects are still not well understood. In this study, we find that TSPs suppressed inflammatory cytokines (TNF and IL-6) in Mɸs stimulated with a broad panel of TLR agonists, whilst inducing IL-10. Moreover, M1 markers such as MHCII, CD86, iNOS, and TNF were downregulated in TSP-treated Mɸs, without polarizing them towards an M2-like phenotype. We showed that TSPs could establish a suppressed activation state of Mɸs lasting at least for 72 h, indicating an anti-inflammatory innate training. Moreover, we found that TSPs, via repression of intracellular TNF generation, decreased its secretion rather than interfering with the release of surface-bound TNF. Metabolic analysis showed that TSPs promote oxidative phosphorylation (OXPHOS) without affecting glycolytic rate. Collectively, these findings expand our knowledge on helminth-induced immune modulation and support future investigations into the anti-inflammatory properties of TSPs for therapeutic purposes.

The neglected tropical disease trichuriasis is caused by the whipworm Trichuris trichiura, a soil-transmitted helminth that has infected humans for millennia. Today, T. trichiura infects as many as 500 million people, predominantly in communities with poor sanitary infrastructure enabling sustained faecal-oral transmission. Using whole-genome sequencing of geographically distributed worms collected from human and other primate hosts, together with ancient samples preserved in archaeologically-defined latrines and deposits dated up to one thousand years old, we present the first population genomics study of T. trichiura. We describe the continent-scale genetic structure between whipworms infecting humans and baboons relative to those infecting other primates. Admixture and population demographic analyses support a stepwise distribution of genetic variation that is highest in Uganda, consistent with an African origin and subsequent translocation with human migration. Finally, genome-wide analyses between human samples and between human and non-human primate samples reveal local regions of genetic differentiation between geographically distinct populations. These data provide insight into zoonotic reservoirs of human-infective T. trichiura and will support future efforts toward the implementation of genomic epidemiology of this globally important helminth.

Regulation of macrophage (Mɸ) function can maintain tissue homeostasis and control inflammation. Parasitic worms (helminths) are potent modulators of host immune and inflammatory responses. They have evolved various strategies to promote immunosuppression, including redirecting phagocytic cells toward a regulatory phenotype. Although soluble products from the whipworm Trichuris suis (TSPs) have shown significant effects on Mɸ function, the mechanisms underlying these modulatory effects are still not well understood. In this study, we find that TSPs suppressed inflammatory cytokines (TNF and IL-6) in Mɸs stimulated with a broad panel of TLR agonists, whilst inducing IL-10. Moreover, M1 markers such as MHCII, CD86, iNOS, and TNF were downregulated in TSP-treated Mɸs, without polarizing them towards an M2-like phenotype. We showed that TSPs could establish a suppressed activation state of Mɸs lasting at least for 72 h, indicating an anti-inflammatory innate training. Moreover, we found that TSPs, via repression of intracellular TNF generation, decreased its secretion rather than interfering with the release of surface-bound TNF. Metabolic analysis showed that TSPs promote oxidative phosphorylation (OXPHOS) without affecting glycolytic rate. Collectively, these findings expand our knowledge on helminth-induced immune modulation and support future investigations into the anti-inflammatory properties of TSPs for therapeutic purposes.

Dietary probiotics may enhance gut health by directly competing with pathogenic agents and through immunostimulatory effects. These properties are recognized in the context of bacterial and viral pathogens, but less is known about interactions with eukaryotic pathogens such as parasitic worms (helminths). In this study we investigated whether two probiotic mixtures (comprised of Bacillus amyloliquefaciens, B. subtilis, and Enterococcus faecium [BBE], or Lactobacillus rhamnosus LGG and Bifidobacterium animalis subspecies Lactis Bb12 [LB]) could modulate helminth infection kinetics as well as the gut microbiome and intestinal immune responses in pigs infected with the nodular worm Oesophagostomum dentatum. We observed that neither probiotic mixture influenced helminth infection levels. BBE, and to a lesser extent LB, changed the alpha- and beta-diversity indices of the colon and fecal microbiota, notably including an enrichment of fecal Bifidobacterium spp. by BBE. However, these effects were muted by concurrent O. dentatum infection. BBE (but not LB) significantly attenuated the O. dentatum-induced upregulation of genes involved in type-2 inflammation and restored normal lymphocyte ratios in the ileo-caecal lymph nodes that were altered by infection. Moreover, inflammatory cytokine release from blood mononuclear cells and intestinal lymphocytes was diminished by BBE. Collectively, our data suggest that selected probiotic mixtures can play a role in maintaining immune homeostasis during type 2-biased inflammation. In addition, potentially beneficial changes in the microbiome induced by dietary probiotics may be counteracted by helminths, highlighting the complex inter-relationships that potentially exist between probiotic bacteria and intestinal parasites.

Dietary probiotics may enhance gut health by directly competing with pathogenic agents and through immunostimulatory effects. These properties are recognized in the context of bacterial and viral pathogens, but less is known about interactions with eukaryotic pathogens such as parasitic worms (helminths). In this study we investigated whether two probiotic mixtures (comprised of Bacillus amyloliquefaciens, B. subtilis, and Enterococcus faecium [BBE], or Lactobacillus rhamnosus LGG and Bifidobacterium animalis subspecies Lactis Bb12 [LB]) could modulate helminth infection kinetics as well as the gut microbiome and intestinal immune responses in pigs infected with the nodular worm Oesophagostomum dentatum. We observed that neither probiotic mixture influenced helminth infection levels. BBE, and to a lesser extent LB, changed the alpha- and beta-diversity indices of the colon and fecal microbiota, notably including an enrichment of fecal Bifidobacterium spp. by BBE. However, these effects were muted by concurrent O. dentatum infection. BBE (but not LB) significantly attenuated the O. dentatum-induced upregulation of genes involved in type-2 inflammation and restored normal lymphocyte ratios in the ileo-caecal lymph nodes that were altered by infection. Moreover, inflammatory cytokine release from blood mononuclear cells and intestinal lymphocytes was diminished by BBE. Collectively, our data suggest that selected probiotic mixtures can play a role in maintaining immune homeostasis during type 2-biased inflammation. In addition, potentially beneficial changes in the microbiome induced by dietary probiotics may be counteracted by helminths, highlighting the complex inter-relationships that potentially exist between probiotic bacteria and intestinal parasites.

Dietary probiotics may enhance gut health by directly competing with pathogenic agents and through immunostimulatory effects. These properties are recognized in the context of bacterial and viral pathogens, but less is known about interactions with eukaryotic pathogens such as parasitic worms (helminths). In this study we investigated whether two probiotic mixtures (comprised of Bacillus amyloliquefaciens, B. subtilis, and Enterococcus faecium [BBE], or Lactobacillus rhamnosus LGG and Bifidobacterium animalis subspecies Lactis Bb12 [LB]) could modulate helminth infection kinetics as well as the gut microbiome and intestinal immune responses in pigs infected with the nodular worm Oesophagostomum dentatum. We observed that neither probiotic mixture influenced helminth infection levels. BBE, and to a lesser extent LB, changed the alpha- and beta-diversity indices of the colon and fecal microbiota, notably including an enrichment of fecal Bifidobacterium spp. by BBE. However, these effects were muted by concurrent O. dentatum infection. BBE (but not LB) significantly attenuated the O. dentatum-induced upregulation of genes involved in type-2 inflammation and restored normal lymphocyte ratios in the ileo-caecal lymph nodes that were altered by infection. Moreover, inflammatory cytokine release from blood mononuclear cells and intestinal lymphocytes was diminished by BBE. Collectively, our data suggest that selected probiotic mixtures can play a role in maintaining immune homeostasis during type 2-biased inflammation. In addition, potentially beneficial changes in the microbiome induced by dietary probiotics may be counteracted by helminths, highlighting the complex inter-relationships that potentially exist between probiotic bacteria and intestinal parasites.

A Corrigendum on Augmented COlorimetric NANoplasmonic (CONAN) Method for Grading Purity and Determine Concentration of EV Microliter Volume Solutions by Zendrini, A., Paolini, L., Busatto, S., Radeghieri, A., Romano, M., Wauben, M. H. M., et al. (2020). Front. Bioeng. Biotechnol. 7:452. doi: 10.3389/fbioe.2019.00452

Emerging evidence suggests that immune cells not only communicate with each other through cytokines, chemokines, and cell surface receptors, but also by releasing small membranous structures known as extracellular vesicles (EVs). EVs carry a variety of different molecules that can be taken up by recipient cells. Parasitic worms are well known for their immunomodulatory properties, but whether they can affect immune responses by altering EV-driven communication between host immune cells remains unclear. Here we provide evidence that stimulation of bone marrow-derived macrophages (BMDMs) with soluble products of Trichuris suis (TSPs), leads to the release of EVs with anti-inflammatory properties. Specifically, we found that EVs from TSP-pulsed BMDMs, but not those from unstimulated BMDMs can suppress TNFα and IL-6 release in LPS-stimulated BMDMs and BMDCs. However, no polarization toward M1 or M2 was observed in macrophages exposed to EVs. Moreover, EVs enhanced reactive oxygen species (ROS) production in the exposed BMDMs, which was associated with a deregulated redox homeostasis as revealed by pathway analysis of transcriptomic data. Proteomic analysis identified cytochrome p450 (CYP450) as a potential source of ROS in EVs from TSP-pulsed BMDMs. Finally, pharmacological inhibition of CYP450 activity could suppress ROS production in those BMDMs. In summary, we find that TSPs can modulate immune responses not only via direct interactions but also indirectly by eliciting the release of EVs from BMDMs that exert anti-inflammatory effects on recipient cells.

Increasing evidence suggests that nutritional manipulation of the commensal gut microbiota (GM) may play a key role in maintaining animal health and production in an era of reduced antimicrobial usage. Gastrointestinal helminth infections impose a considerable burden on animal performance, and recent studies suggest that infection may substantially alter the composition and function of the GM. Here, we discuss the potential interactions between different bioactive dietary components (prebiotics, probiotics and phytonutrients) and helminth infection on the GM in livestock. A number of recent studies suggest that host diet can strongly influence the nature of the helminth-GM interaction. Nutritional manipulation of the GM may thus impact helminth infection, and conversely infection may also influence how the GM responds to dietary interventions. Moreover, a dynamic interaction exists between helminths, the GM, intestinal immune responses, and inflammation. Deciphering the mechanisms underlying the diet-GM-helminth axis will likely inform future helminth control strategies, as well as having implications for how health-promoting feed additives, such as probiotics, can play a role in sustainable animal production.

Increasing evidence suggests that nutritional manipulation of the commensal gut microbiota (GM) may play a key role in maintaining animal health and production in an era of reduced antimicrobial usage. Gastrointestinal helminth infections impose a considerable burden on animal performance, and recent studies suggest that infection may substantially alter the composition and function of the GM. Here, we discuss the potential interactions between different bioactive dietary components (prebiotics, probiotics and phytonutrients) and helminth infection on the GM in livestock. A number of recent studies suggest that host diet can strongly influence the nature of the helminth-GM interaction. Nutritional manipulation of the GM may thus impact helminth infection, and conversely infection may also influence how the GM responds to dietary interventions. Moreover, a dynamic interaction exists between helminths, the GM, intestinal immune responses, and inflammation. Deciphering the mechanisms underlying the diet-GM-helminth axis will likely inform future helminth control strategies, as well as having implications for how health-promoting feed additives, such as probiotics, can play a role in sustainable animal production.

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

Trichuris trichiura and T. suis are whipworms of humans and pigs, respectively, but it has recently been suggested that humans may be infected with multiple genotypes or species of Trichuris and cross-infection with Trichuris of pig origin has also been reported. In addition, the species status of Trichuris in non-human primates is unsettled and it is unknown how many whipworm species we share with other primates. Herein, we inferred the phylogeny of Trichuris collected from human, baboon and pig based on nuclear (18S and beta-tubulin) and mitochondrial (cox1) genes and evaluated the use of three PCR linked restriction fragment length polymorphism (PCR-RFLP) to identify worms. We found that all baboon worms clustered with human worms and that all these primate worms are different from T. suis. In general, there was an agreement between the phylogeny established based on the nuclear and mtDNA genes. However, we found evidence for non-targeted cox1 gene amplification for a subset of the human worms and suggest the presence of mitochondrial pseudogenes (numts) of pig cox1 gene in the human Trichuris genome. In conclusion, phylogenetic characterization of human whipworm based on the cox1 gene alone may be problematic without suitable preceded measures to avoid the numts amplification.

Toxocara and Toxascaris are parasitic nematodes that infect canids and felids although species of the genus Toxocara also infect humans. This work aimed to establish the phylogenetic and phylogeographic relationship between specimens of T. canis, T. cati, T. malaysiensis and Toxascaris leonina and to evaluate the degree of host specificity. In total, 437 samples (adults and pools of eggs) were collected from canids and felids from eight countries. Parasites were identified by morphology, PCR linked Restriction Fragment Length Polymorphism (PCR-RFLP) and partial sequencing of the mitochondrial gene cox1. Phylogenetic trees were constructed and genetic distance among isolates was estimated. Based on the molecular characterization all worms were identified in agreement with their respective hosts with the exception of three samples; two from cats and one from dogs identified as T. canis and T. cati, respectively. There was no clear geographical clustering of the samples despite this study including parasites from three continents. This is the first study, to our knowledge, to use molecular methods to identify T. canis in cats and T. cati in dogs with host specificity being the most common finding. Our developed PCR-RFLP method was found to be a facile and reliable method for identifying Toxocara species.

The gut microbiota (GM) displays a profound ability to adapt to extrinsic factors, such as gastrointestinal pathogens and/or dietary alterations. Parasitic worms (helminths) and host-associated GM share a long co-evolutionary relationship, exerting mutually modulatory effects which may impact the health of the host. Moreover, dietary components such as prebiotic fibers (e.g. inulin) are capable of modulating microbiota toward a composition often associated with a healthier gut function. The effect of helminth infection on the host microbiota is still equivocal, and it is also unclear how parasites and prebiotic dietary components interact to influence the microbiota and host health status. Some helminths, such as Trichuris suis (porcine whipworm), also exhibit strong immunomodulatory and anti-inflammatory effects. We therefore explored the effects of T. suis, alone and in interaction with inulin, both in fecal microbiota during the infection period and luminal microbiota across four intestinal segments at the end of a 4-week infection period. We observed that T. suis generally had minimal, but mainly positive, effects on the microbiota. T. suis increased the relative abundance of bacterial genera putatively associated with gut health such as Prevotella, and decreased bacteria such as Proteobacteria that have been associated with dysbiosis. Interestingly, dietary inulin interacted with T. suis to enhance these effects, thereby modulating the microbiota toward a composition associated with reduced inflammation. Our results show that administration of T. suis together with the consumption of prebiotic inulin may have the potential to positively affect gut health.

The soil transmitted whipworm, Trichuris trichiura affects about half a billion people globally. Diagnosis is based on identification of characteristic lemon-shaped eggs in stool samples. Occasionally large Trichuris eggs have been reported and have been ascribed to variation within T. trichiura or zoonotic infection with T. vulpis from dogs. We observed that the egg size profile changed markedly after anthelmintic treatment, and remained so, in a human individual self-infected with T. trichiura. The large eggs were detected with two standard diagnostic methods, Kato-Katz thick smear and salt-flotation (McMaster) method. It is therefore important to bear in mind that the morphology of parasite eggs may vary, e.g. in response to previous drug treatment. Large Trichuris eggs in human stool samples should not be diagnosed as T. vulpis infection without confirmation by other means.

Toxocara and Toxascaris are parasitic nematodes that infect canids and felids although species of the genus Toxocara also infect humans. This work aimed to establish the phylogenetic and phylogeographic relationship between specimens of T. canis, T. cati, T. malaysiensis and Toxascaris leonina and to evaluate the degree of host specificity. In total, 437 samples (adults and pools of eggs) were collected from canids and felids from eight countries. Parasites were identified by morphology, PCR linked Restriction Fragment Length Polymorphism (PCR-RFLP) and partial sequencing of the mitochondrial gene cox1. Phylogenetic trees were constructed and genetic distance among isolates was estimated. Based on the molecular characterization all worms were identified in agreement with their respective hosts with the exception of three samples; two from cats and one from dogs identified as T. canis and T. cati, respectively. There was no clear geographical clustering of the samples despite this study including parasites from three continents. This is the first study, to our knowledge, to use molecular methods to identify T. canis in cats and T. cati in dogs with host specificity being the most common finding. Our developed PCR-RFLP method was found to be a facile and reliable method for identifying Toxocara species.

The gut microbiota (GM) displays a profound ability to adapt to extrinsic factors, such as gastrointestinal pathogens and/or dietary alterations. Parasitic worms (helminths) and host-associated GM share a long co-evolutionary relationship, exerting mutually modulatory effects which may impact the health of the host. Moreover, dietary components such as prebiotic fibers (e.g. inulin) are capable of modulating microbiota toward a composition often associated with a healthier gut function. The effect of helminth infection on the host microbiota is still equivocal, and it is also unclear how parasites and prebiotic dietary components interact to influence the microbiota and host health status. Some helminths, such as Trichuris suis (porcine whipworm), also exhibit strong immunomodulatory and anti-inflammatory effects. We therefore explored the effects of T. suis, alone and in interaction with inulin, both in fecal microbiota during the infection period and luminal microbiota across four intestinal segments at the end of a 4-week infection period. We observed that T. suis generally had minimal, but mainly positive, effects on the microbiota. T. suis increased the relative abundance of bacterial genera putatively associated with gut health such as Prevotella, and decreased bacteria such as Proteobacteria that have been associated with dysbiosis. Interestingly, dietary inulin interacted with T. suis to enhance these effects, thereby modulating the microbiota toward a composition associated with reduced inflammation. Our results show that administration of T. suis together with the consumption of prebiotic inulin may have the potential to positively affect gut health.

Helminth parasites have a remarkable ability to persist within their mammalian hosts, which is largely due to their secretion of molecules with immunomodulatory properties. Although the soluble components of helminth secretions have been extensively studied, the discovery that helminths release extracellular vesicles (EVs) has added further complexity to the host-parasite interaction. Whilst several studies have begun to characterise the molecules carried by helminth EVs, work aimed at investigating their biological functions has been hindered by a lack of helminth-specific EV markers. To begin to address this, we summarised helminth EV literature to date. With a focus on the protein and microRNA (miRNA) cargo, we aimed to detect similarities and differences across those major groups of helminths for which data are available; namely nematodes, trematodes and cestodes. Pfam analysis revealed that although there is no universal EV marker for all helminth species, the EF-hand protein family was present in all EV datasets from cestodes and trematodes, and could serve as a platyhelminth EV biomarker. In contrast, M13 metallopeptidases and actin may have potential as markers for nematode EVs. As with proteins, many miRNA families appeared to be species-, stage-, or dataset-specific. Two miRNA families were common to nematode EVs (mir-10 and let-7); the miRNA cargo of EVs secreted by clade I species appeared somewhat different from species from other clades. Five miRNA families (mir-71, mir-10, mir-190, let-7 and mir-2) were shared by all trematode species examined. Our analysis has identified novel markers that may be used in studies aimed at characterising helminth EVs and interrogating their function at the host-parasite interface. In addition, we discuss the heterogeneity of methods used for helminth EV isolation and emphasise the need for a standardised approach in reporting on helminth EV data.

We herein describe an Atomic Force Microscopy (AFM)-based experimental procedure which allows the simultaneous mechanical and morphological characterization of several hundred individual nanosized vesicles within the hour timescale. When deposited on a flat rigid surface from aqueous solution, vesicles are deformed by adhesion forces into oblate spheroids whose geometry is a direct consequence of their mechanical stiffness. AFM image analysis can be used to quantitatively measure the contact angle of individual vesicles, which is a size-independent descriptor of their deformation and, consequently, of their stiffness. The same geometrical measurements can be used to infer vesicle diameter in its original, spherical shape. We demonstrate the applicability of the proposed approach to natural vesicles obtained from different sources, recovering their size and stiffness distributions by simple AFM imaging in liquid. We show how the combined EV stiffness/size readout is able to discriminate between subpopulations of vesicular and non-vesicular objects in the same sample, and between populations of vesicles with similar sizes but different mechanical characteristics. We also discuss a force spectroscopy calibration procedure to quantitatively link the stiffness of EVs to their average contact angle. Finally, we discuss expected extensions and applications of the methodology.

The prevalent porcine helminth, Ascaris suum, compromises pig health and reduces farm productivity worldwide. The closely related human parasite, A. lumbricoides, infects more than 800 million people representing a disease burden of 1.31 million disability-adjusted life years. The infections are often chronic in nature, and the parasites have a profound ability to modulate their hosts' immune responses. This study provides the first in-depth characterisation of extracellular vesicles (EVs) from different developmental stages and body parts of A. suum and proposes the role of these vesicles in the host-parasite interplay. The release of EVs from the third- (L3) and fourth-stage (L4) larvae and adults was demonstrated by transmission electron microscopy (TEM), and sequencing of EV-derived RNA identified a number of microRNAs (miRNAs) and transcripts of potential host immune targets, such as IL-13, IL-25 and IL-33, were identified. Furthermore, proteomics of EVs identified several proteins with immunomodulatory properties and other proteins previously shown to be associated with parasite EVs. Taken together, these results suggest that A. suum EVs and their cargo may play a role in host-parasite interactions. This knowledge may pave the way to novel strategies for helminth infection control and knowledge of their immune modulatory potential.