A complete proteomic profile of human and bovine milk exosomes by liquid chromatography mass spectrometry.

BACKGROUND: The present study investigates the proteomic content of milk-derived exosomes. A detailed description of the content of milk exosomes is essential to improve our understanding of the various components of milk and their role in nutrition. METHODS: The exosomes used in this study were isolated as previously described and characterized by their morphology, particle concentration, and the presence of exosomal markers. Human and bovine milk exosomes were evaluated using Information-Dependent Acquisition (IDA) Mass Spectrometry. A direct comparison is made between their proteomic profiles. RESULTS: IDA analyses revealed similarities and differences in protein content. About 229 and 239 proteins were identified in the human and bovine milk exosome proteome, respectively, of which 176 and 186 were unique to each species. Fifty-three proteins were common in both groups. These included proteins associated with specific biological processes and molecular functions. Most notably, the 4 abundant milk proteins lactadherin, butyrophilin, perilipin-2, and xanthine dehydrogenase/oxidase were present in the top 20 list for both human and bovine milk exosomes. CONCLUSION: The milk exosome protein profiles we have provided are crucial new information for the field of infant nutrition. They provide new insight into the components of milk from both humans and bovines.

Phenotypic, functional, and plasticity features of classical and alternatively activated human macrophages.

Macrophages are dynamic cells that mature under the influence of signals from the local microenvironment into either classically (M1) or alternatively (M2) activated macrophages with specific functional and phenotypic properties. Although the phenotypic identification of M1 and M2 macrophages is well established in mice, this is less clear for human macrophages. In addition, the persistence and reversibility of polarized human phenotypes is not well established. Human peripheral blood monocytes were differentiated into uncommitted macrophages (M0) and then polarized to M1 and M2 phenotypes using LPS/IFN-γ and IL-4/IL-13, respectively. M1 and M2 were identified as CD64(+)CD80(+) and CD11b(+)CD209(+), respectively, by flow cytometry. Polarized M1 cells secreted IP-10, IFN-γ, IL-8, TNF-α, IL-1ß, and RANTES, whereas M2 cells secreted IL-13, CCL17, and CCL18. Functionally, M2 cells were highly endocytic. In cytokine-deficient medium, the polarized macrophages reverted back to the M0 state within 12 days. If previously polarized macrophages were given the alternative polarizing stimulus after 6 days of resting in cytokine-deficient medium, a switch in polarization was seen (i.e., M1 macrophages switched to M2 and expressed CD11b(+)CD209(+) and vice versa). In summary, we report phenotypic identification of human M1 and M2 macrophages, their functional characteristics, and their ability to be reprogrammed given the appropriate stimuli.

The exosome: a review of current therapeutic roles and capabilities in human reproduction.

Exosomes are nano-vesicles (30-150 nm) which may be useful as therapeutic delivery vehicles and as diagnostic biomarkers. Exosomes are produced naturally within the human body and therefore are not prone to immunogenicity effects which would otherwise destroy unelicited foreign bodies. Clinically, they have been regarded as ideal candidates for applications relating to biomarker developments for the early detection of different diseases. Furthermore, exosomes may be of interest as potential drug delivery vehicles, which may improve factors such as bioavailability of loaded molecular cargo, side effect profiles, off-target effects, and pharmacokinetics of drug molecules. In this review, the therapeutic potential of exosomes and their use as clinical biomarkers for early diagnostics will be explored, alongside exosomes as therapeutic delivery vehicles. This review will evaluate techniques for cargo loading, and the capacity of loaded exosomes to improve various reproductive disease states. It becomes important, therefore, to consider factors such as loading efficiency, loading methods, cell viability, exosomal sources, exosome isolation, and the potential therapeutic benefits of exosomes. Issues related to targeted drug delivery will also be discussed. Finally, the variety of therapeutic cargo and the application of appropriate loading methods is explored, in the context of establishing clinical utility. Exosomes have more recently been widely accpeted as potential tools for disease diagnostics and the targeted delivery of certain therapeutic molecules-and in due time exosomes will be utilised more commonly within the clinical setting. Specifically, exosomal biomarkers can be identified and related to various detrimental conditions which occur during pregnancy. Considering, this review will explore the potential future of exosomes as both diagnostic tools and therapeutic delivery vehicles to treat related conditions, including the challenges which exist towards incorporating exosomes within the clinical environment to benefit patients.

Dynamics of inflammatory cytokine expression in bovine endometrial cells exposed to cow blood plasma small extracellular vesicles (sEV) may reflect high fertility.

Aberrant inflammation in the endometrium impairs reproduction and leads to poor fertility. Small extracellular vesicles (sEV) are nanoparticles 30-200 nm in-size and contain transferable bioactive molecules that reflect the parent cell. Holstein-Friesian dairy cows with divergent genetic merit, high- (n = 10) and low-fertile (n = 10), were identified based on fertility breeding value (FBV), cow ovulation synchronization and postpartum anovulatory intervals (PPAI). In this study, we evaluated the effects of sEVs enriched from plasma of high-fertile (HF-EXO) and low-fertile (LF-EXO) dairy cows on inflammatory mediator expression by bovine endometrial epithelial (bEEL) and stromal (bCSC) cells. Exposure to HF-EXO in bCSC and bEEL cells yielded lower expression of PTGS1 and PTGS2 compared to the control. In bCSC cells exposed to HF-EXO, pro-inflammatory cytokine IL1-α was downregulated compared to the untreated control, IL-12α and IL-8 were downregulated compared to the LF-EXO treatment. Our findings demonstrate that sEVs interact with both endometrial epithelial and stromal cells to initiate differential gene expression, specifically genes relate to inflammation. Therefore, even subtle changes on the inflammatory gene cascade in the endometrium via sEV may affect reproductive performance and/or outcomes. Further, sEV from high-fertile animals acts in a unique direction to deactivate prostaglandin synthases in both bCSC and bEEL cells and deactivate pro-inflammatory cytokines in the endometrial stroma. The results suggest that circulating sEV may serve as a potential biomarker of fertility.

International Survey of Specialist Fetal Alcohol Spectrum Disorder Diagnostic Clinics: Comparison of Diagnostic Approach and Considerations Regarding the Potential for Unification.

Fetal alcohol spectrum disorder (FASD) is a prevalent neurodevelopmental condition. Despite FASD being recognized as a clinical disorder there is no globally agreed set of diagnostic criteria. Accurate and timely diagnosis of FASD is imperative to inform clinical care, optimize outcomes for individuals accessing assessments and their families, as well as for research and prevention strategies. To inform movement towards a unified approach, the present study aimed to capture an international perspective on current FASD diagnostic criteria, as well as potential barriers and facilitators to unification. An online survey was created using REDCap and sent to clinics identified and contacted via internet searches. Quantitative data were presented using descriptive statistics and open-ended questions analysed using content analysis. The survey captured information about each clinic's current diagnostic approach, whether they would support a unified method, and the barriers and facilitators for a consistent international FASD diagnostic approach. Fifty-five (37.4%) of 147 FASD clinics identified worldwide participated. The majority (n = 50, 90.9%) of respondents supported a unified approach. Content analysis identified a lack of collaboration as a key barrier, while strong leadership in guideline creation and implementation emerged as a central facilitator. These barriers and facilitators can be used to guide future collaborative efforts towards implementing consistent diagnostic criteria.

Neutrophil respiratory burst activity is not exaggerated in cystic fibrosis.

BACKGROUND: Exaggerated neutrophil-dominated inflammation underlies progressive cystic fibrosis (CF) lung disease. Older studies reported a defective respiratory burst in CF, but more recent studies suggest neutrophil function is normal. METHODS: We measured the amount and rate of reactive oxygen species (ROS) during PMA-stimulated respiratory burst activity in children [70 CF, 13 disease controls, 19 health controls] and adults [31 CF, 14 health controls] in neutrophils harvested from peripheral blood. Blood was collected from participants with CF when clinically stable (60 children, 9 adults) and on hospital admission (38 children, 24 adults) and discharge (18 children, 21 adults) for acute pulmonary exacerbations. RESULTS: When clinically stable, children with CF had lower ROS production [median 318,633, 25% 136,810 - 75% 569,523 RLU] than disease controls [median 599,459, 25% 425,566 - 75% 730,527 RLU] and healthy controls [median 534,073, 25% 334,057 - 75% 738,593 RLU] (p = 0.008). The rate of ROS production was also lower (p = 0.029). In neither children nor adults with CF did ROS production increase on hospital admission for acute pulmonary exacerbation, nor fall prior to discharge. There were no associations between ROS production and high-sensitivity C-reactive protein (indicating systemic inflammation) in either children or adults with CF. CONCLUSIONS: Our data do not support a role for exaggerated respiratory burst activity contributing to the exaggerated neutrophil-dominated inflammation seen with CF lung disease.

Proteomic profiling of plasma-derived small extracellular vesicles: a novel tool for understanding the systemic effects of tick burden in cattle.

Cattle ticks pose a significant threat to the health and profitability of cattle herds globally. The investigation of factors leading to natural tick resistance in cattle is directed toward targeted breeding strategies that may combat cattle tick infestation on the genetic level. Exosomes (EXs), small extracellular vesicles (EVs) of 50 to 150 nm diameter, are released from all cell types into biofluids such as blood plasma and milk, have been successfully used in diagnostic and prognostic studies in humans, and can provide essential information regarding the overall health state of animals. Mass spectrometry (MS) is a highly sensitive proteomics application that can be used to identify proteins in a complex mixture and is particularly useful for biomarker development. In this proof of principle study, EXs were isolated from the blood plasma of cattle (Bos taurus) with high (HTR) and low tick resistance (LTR) (n = 3/group). Cattle were classified as HTR or LTR using a tick scoring system, and EXs isolated from the cattle blood plasma using an established protocol. EXs were subjected to MS analysis in data-dependent acquisition mode and protein search performed using Protein Pilot against the B. taurus proteome. A total of 490 unique proteins were identified across all samples. Of these, proteins present in all replicates from each group were selected for further analysis (HTR = 121; LTR = 130). Gene ontology analysis was performed using PANTHER GO online software tool. Proteins unique to HTR and LTR cattle were divided by protein class, of which 50% were associated with immunity/defense in the HTR group, whereas this protein class was not detected in EXs from LTR cattle. Similarly, unique proteins in HTR cattle were associated with B-cell activation, immunoglobins, immune response, and cellular iron ion homeostasis. In LTR cattle, unique exosomal proteins were associated with actin filament binding, purine nucleotide binding, plasma membrane protein complex, and carbohydrate derivative binding. This is the first study to demonstrate that MS analysis of EXs derived from the blood plasma of HTR and LTR cattle can be successfully applied to profile the systemic effects of tick burden.

Cattle ticks are a significant burden to cattle industries globally. Current methods to treat cattle ticks are costly and inefficient in the long term. It has been noted that while some cattle may exhibit a natural resistance to ticks, others carry a heavy tick burden. The study of small extracellular vesicles, or exosomes (EXs), isolated from cattle blood plasma provides a noninvasive way of analyzing changes at the cellular level and may be of use in understanding the systemic effects of tick burden or factors leading to natural resistance. The aim of this study was to assess high (HTR) and low tick resistance (LTR) cattle identified using a tick burden scoring system by analyzing the protein content of circulating EXs via qualitative proteomics analysis. We found that a class of proteins related to defense/immunity comprised 50% of proteins unique to HTR cattle, while this protein class was not detected in proteins unique to LTR cattle. Additionally, epidermal growth factor­calcium-binding protein domains were 2-fold increased in LTR cattle compared with HTR cattle, indicating a possible mechanism for widespread metabolic change. This is the first study to employ proteomic analysis of exosomal cargo as an approach to understanding the systemic effects of tick burden in cattle.

A Comparison of Blood Plasma Small Extracellular Vesicle Enrichment Strategies for Proteomic Analysis.

Proteomic analysis of small extracellular vesicles (sEVs) poses a significant challenge. A 'gold-standard' method for plasma sEV enrichment for downstream proteomic analysis is yet to be established. Methods were evaluated for their capacity to successfully isolate and enrich sEVs from plasma, minimise the presence of highly abundant plasma proteins, and result in the optimum representation of sEV proteins by liquid chromatography tandem mass spectrometry. Plasma from four cattle (Bos taurus) of similar physical attributes and genetics were used. Three methods of sEV enrichment were utilised: ultracentrifugation (UC), size-exclusion chromatography (SEC), and ultrafiltration (UF). These methods were combined to create four groups for methodological evaluation: UC + SEC, UC + SEC + UF, SEC + UC and SEC + UF. The UC + SEC method yielded the highest number of protein identifications (IDs). The SEC + UC method reduced plasma protein IDs compared to the other methods, but also resulted in the lowest number of protein IDs overall. The UC + SEC + UF method decreased sEV protein ID, particle number, mean and mode particle size, particle yield, and did not improve purity compared to the UC + SEC method. In this study, the UC + SEC method was the best method for sEV protein ID, purity, and overall particle yield. Our data suggest that the method and sequence of sEV enrichment strategy impacts protein ID, which may influence the outcome of biomarker discovery studies.

Characterisation of ReNcells CX and VM stimulated with interleukin-1ß and lipopolysaccharide.

Abnormal and dysregulated neuroinflammation has been linked to many neurological disorders and neurodegenerative diseases. Understanding the mechanisms of neuroinflammation, their impact on neurodevelopment and how neuroinflammation might be modulated, are currently considered to be critical to improving neurological treatment. ReNcell CX (originating from the cortical region) and VM (originating from the ventral mesencephalon) are human immortalised neural stem cell lines, that have the potential to be used as experimental models for investigating neuroinflammation in vitro. However, the information on the inflammation response of these cells is limited. This is especially more so for undifferentiated ReNcells. In this report we demonstrate using ELISA that cultured, undifferentiated ReNcell CX and VM produce significant amounts of IL-6 in response to IL-1ß treatment, but not to LPS treatment. Additionally, conventional RT-PCR showed that ReNcell CX cells expressed TNFR1 and NF-κB, whereas ReNcell VM expressed only NF-κB. Our results encourage further investigation into the relationship between 1L-1ß and IL-6 in both ReNcell CX and VM. Moreover, TNF-α treatment might potentially affect neuroinflammation in ReNcell CX, while activation of the NF-κB pathway could also play a critical part in neuroinflammation.

Differentially Expressed Extracellular Vesicle, Exosome and Non-Exosome miRNA Profile in High and Low Tick-Resistant Beef Cattle.

Heavy tick burden on beef cattle account for huge economic losses globally, with an estimated value of US$22-30 billion per annum. In Australia, ticks cost the northern beef industry approximately A$170-200 million. Methods to evaluate and predict tick resistance would therefore be of great value to the global cattle trade. Exosomes (EX) are small extracellular vesicles (EVs) of ~30-150nm diameter and have gained popularity for their diagnostic and prognostic potential. EX contain, among other biomolecules, various types of RNA including micro-RNA (miRNA) and long noncoding RNA (lncRNA). MiRNA specifically have been validated as therapeutic biomarkers as they perform regulatory functions at the post-transcriptional level and are differentially expressed between divergent groups. The objective of the present study was to evaluate the miRNA profiles of EV and fractionated exosomal samples of high and low tick-resistant beef cattle to highlight potential miRNA biomarkers of tick resistance. Cows (n = 3/group) were classified into high or low tick resistant groups according to a novel scoring system. EVs and EX were isolated and fractionated from the blood plasma of high and low tick resistant cattle using established isolation and enrichment protocols. The resultant EX and non-EX samples were processed for next generation miRNA sequencing. Offspring of the cows in each high and low tick resistant group underwent the same processing for blood plasma EX, non-EX and miRNA analysis to evaluate the heritability of miRNA associated with tick resistance. A total of 2631 miRNAs were identified in EX and non-EX fractionated samples from high and low tick-resistant beef cattle. MiR-449a was highly expressed in maternal high tick-resistant EX samples. Of these, 174 were novel miRNAs, and 10 were differentially expressed (DE) (FDR < 0.05). These 10 DE miRNAs were also present in EVs, and three miRNAs were highly expressed: miR-2419-3p, miR-7861-3p and miR-2372-5p. Although 196 novel miRNAs were identified in fractionated samples of offspring, no miRNA were differentially expressed in these animals.

Comprehensive analysis of the secreted proteome of adult Necator americanus hookworms.

The human hookworm Necator americanus infects more than 400 million people worldwide, contributing substantially to the poverty in these regions. Adult stage N. americanus live in the small intestine of the human host where they inject excretory/secretory (ES) products into the mucosa. ES products have been characterized at the proteome level for a number of animal hookworm species, but until now, the difficulty in obtaining sufficient live N. americanus has been an obstacle in characterizing the secretome of this important human pathogen. Herein we describe the ES proteome of N. americanus and utilize this information along with RNA Seq data to conduct the first proteogenomic analysis of a parasitic helminth, significantly improving the available genome and thereby generating a robust description of the parasite secretome. The genome annotation resulted in a revised prediction of 3,425 fewer genes than initially reported, accompanied by a significant increase in the number of exons and introns, total gene length and the percentage of the genome covered by genes. Almost 200 ES proteins were identified by LC-MS/MS with SCP/TAPS proteins, 'hypothetical' proteins and proteases among the most abundant families. These proteins were compared to commonly used model species of human parasitic infections, including Ancylostoma caninum, Nippostrongylus brasiliensis and Heligmosomoides polygyrus. SCP/TAPS proteins are immunogenic in nematode infections, so we expressed four of those identified in this study in recombinant form and showed that they are all recognized to varying degrees by serum antibodies from hookworm-infected subjects from a disease-endemic area of Brazil. Our findings provide valuable information on important families of proteins with both known and unknown functions that could be instrumental in host-parasite interactions, including protein families that might be key for parasite survival in the onslaught of robust immune responses, as well as vaccine and diagnostic targets.

Helminth-induced regulatory T cells and suppression of allergic responses.

Infection with helminths has been associated with lower rates of asthma and other allergic diseases. This has been attributed, in part, to the ability of helminths to induce regulatory T cells that suppress inappropriate immune responses to allergens. Recent compelling evidence suggests that helminths may promote regulatory T cell expansion or effector functions through either direct (secretion of excretory/secretory molecules) or indirect mechanisms (regulation of the microbiome). This review will discuss key findings from human immunoepidemiological observations, studies using animal models of disease, and clinical trials with live worm infections, discussing the therapeutic potential for worms and their secreted products for treating allergic inflammation.

Human Metapneumovirus Impairs Apoptosis of Nasal Epithelial Cells in Asthma via HSP70.

Asthmatics are highly susceptible to respiratory viral infections, possibly due to impaired innate immunity. However, the exact mechanisms of susceptibility are likely to differ amongst viruses. Therefore, we infected primary nasal epithelial cells (NECs) from adults with mild-to-moderate asthma, with respiratory syncytial virus (RSV) or human metapneumovirus (hMPV) in vitro and investigated the antiviral response. NECs from these asthmatics supported elevated hMPV but not RSV infection, compared to non-asthmatic controls. This correlated with reduced apoptosis and reduced activation of caspase-9 and caspase-3/7 in response to hMPV, but not RSV. The expression of heat shock protein 70 (HSP70), a known inhibitor of caspase activation and subsequent apoptosis, was amplified in response to hMPV infection. Chemical inhibition of HSP70 function restored caspase activation and reduced hMPV infection in NECs from asthmatic subjects. There was no impairment in the production of IFN by NECs from asthmatics in response to either hMPV or RSV, demonstrating that increased infection of asthmatic airway cells by hMPV is IFN-independent. This study demonstrates, for the first time, a mechanism for elevated hMPV infection in airway epithelial cells from adult asthmatics and identifies HSP70 as a potential target for antiviral and asthma therapies.

CFTR-dependent defect in alternatively-activated macrophages in cystic fibrosis.

BACKGROUND: The role of the macrophages in cystic fibrosis (CF) lung disease has been poorly studied. We hypothesized that alternatively activated M2 macrophages are abnormal in CF lung disease. METHODS: Blood samples were collected from adults (n=13) children (n=27) with CF on admission for acute pulmonary exacerbation and when clinically stable. Monocytes were differentiated into macrophages and polarized into classical (M1) and alternatively-activated (M2) phenotypes, function determined ex-vivo and compared with healthy controls. RESULTS: In the absence of functional cystic fibrosis trans-membrane conductance regulator (CFTR), either naturally in patients with CF or induced with CFTR inhibitors, monocyte-derived macrophages do not respond to IL-13/IL-4, fail to polarize into M2s associated with a post-transcriptional failure to produce and express IL-13Rα1 on the macrophage surface Polarization to the M1 phenotype was unaffected. CONCLUSIONS: CFTR-dependent imbalance of macrophage phenotypes and functions could contribute to the exaggerated inflammatory response seen in CF lung disease.

Brief exposure to cigarette smoke impairs airway epithelial cell innate anti-viral defence.

BACKGROUND: Human rhinovirus (hRV) infections commonly cause acute upper respiratory infections and asthma exacerbations. Environmental cigarette smoke exposure is associated with a significant increase in the risk for these infections in children. OBJECTIVE: To determine the impact of short-term exposure to cigarette smoke on innate immune responses of airway epithelial cells infected with hRV. METHODS: A human bronchial epithelial cell line (HBEC-3KT) was exposed to cigarette smoke extract (CSE) for 30 min and subsequently infected with hRV serotype 1B. Viral-induced cytokine release was measured with AlphaLISA and viral replication quantified by shed viral titer and intracellular viral copy number 24h post-infection. RESULTS: CSE induced a concentration-dependent decrease in CXCL10 (p<0.001) and IFN-ß (p<0.001), with a 79% reduction at the highest dose with an associated 3-fold increase in shed virus. These effects were maintained when infection was delayed up to 24h post CSE exposure. Exogenous IFN-ß treatment at t=0 after infection blunts the effects of CSE on viral replication (p<0.05). CONCLUSION: A single exposure of 30 min to cigarette smoke has a lasting impact on epithelial innate defence providing a plausible mechanism for the increase in respiratory infections seen in children exposed to second-hand tobacco smoke.