Proteomic profiling of paired human liver homogenate and tissue derived extracellular vesicles.

Advances in technologies to isolate extracellular vesicles (EVs) and detect/quantify their cargo underpin the novel potential of these circulating particles as a liquid biopsy to understand physiology and disease. One organ of particular interest in terms of utilizing EVs as a liquid biopsy is the liver. The extent to which EVs originating from the liver reflect the functional status of this organ remains unknown. This is an important knowledge gap that underpins the utility of circulating liver derived EVs as a liquid biopsy. The primary objective of this study was to characterize the proteomic profile of EVs isolated from the extracellular space of liver tissue (LEV) and compare this profile to that of paired tissue (LH). LCMS analyses detected 2892 proteins in LEV and 2673 in LH. Of the 2673 proteins detected in LH, 1547 (58%) were also detected in LEV. Bioinformatic analyses demonstrated comparable representation of proteins in terms of biological functions and cellular compartments. Although, enriched representation of membrane proteins and associated functions was observed in LEV, while representation of nuclear proteins and associated functions was depleted in LEV. These data support the potential use of circulating liver derived EVs as a liquid biopsy for this organ.

Proteomic characterisation of perhexiline treatment on THP-1 M1 macrophage differentiation.

Background: Dysregulated inflammation is important in the pathogenesis of many diseases including cancer, allergy, and autoimmunity. Macrophage activation and polarisation are commonly involved in the initiation, maintenance and resolution of inflammation. Perhexiline (PHX), an antianginal drug, has been suggested to modulate macrophage function, but the molecular effects of PHX on macrophages are unknown. In this study we investigated the effect of PHX treatment on macrophage activation and polarization and reveal the underlying proteomic changes induced. Methods: We used an established protocol to differentiate human THP-1 monocytes into M1 or M2 macrophages involving three distinct, sequential stages (priming, rest, and differentiation). We examined the effect of PHX treatment at each stage on the polarization into either M1 or M2 macrophages using flow cytometry, quantitative polymerase chain reaction (qPCR) and enzyme linked immunosorbent assay (ELISA). Quantitative changes in the proteome were investigated using data independent acquisition mass spectrometry (DIA MS). Results: PHX treatment promoted M1 macrophage polarization, including increased STAT1 and CCL2 expression and IL-1ß secretion. This effect occurred when PHX was added at the differentiation stage of the M1 cultures. Proteomic profiling of PHX treated M1 cultures identified changes in metabolic (fatty acid metabolism, cholesterol homeostasis and oxidative phosphorylation) and immune signalling (Receptor Tyrosine Kinase, Rho GTPase and interferon) pathways. Conclusion: This is the first study to report on the action of PHX on THP-1 macrophage polarization and the associated changes in the proteome of these cells.

The Nasal Innate Immune Proteome After Saline Irrigation: A Pilot Study in Healthy Individuals.

BACKGROUND: Previous research has shown diminished nasal immune function following nasal saline irrigation (NSI), returning to baseline at 6 hours. The aim of this study was to examine the immune nasal proteome before and after 14 days of nasal irrigation. METHODS: Seventeen healthy volunteers received either isotonic (IsoSal) or low salt (LowNa) NSI. Nasal secretions were collected before and 30 min after NSI at baseline and again after 14 days. Specimens were analyzed using mass spectrometry to detect proteins of relevance to nasal immune function. RESULTS: One thousand eight hundred and sixty-five proteins were identified with significant changes in 71 proteins, of which 23 were identified as part of the innate immune system. Baseline analysis demonstrated an increase of 9 innate proteins after NSI, most after IsoSal. After 14 days, a greater increase in innate peptides was present, with most now in the LowNa group. When NSI solutions were compared, a significant increase in 4 innate proteins, including a 211% in lysozyme, was detected in the LowNa group. CONCLUSION: LowNa NSI demonstrates evidence of improving the innate immune secretions, especially lysozyme, in healthy volunteers.

Oral immunotherapy using boiled peanuts for treating peanut allergy: An open-label, single-arm trial.

BACKGROUND: Peanut allergy affects 1%-3% of children in Western countries. Boiling peanuts has been demonstrated to result in a hypoallergenic product that may provide a safer way of inducing desensitization in peanut-allergic patients by first inducing tolerance to boiled peanut. We aimed to assess the efficacy and safety of oral immunotherapy (OIT) using sequential doses of boiled peanuts followed by roasted peanuts for treating peanut allergy in children. METHODS: In this open-label, phase 2, single-arm clinical trial, children aged 6-18 years with a positive history of peanut allergy and positive peanut skin prick test ≥ 8 mm and/or peanut-specific IgE ≥ 15 kU/L at screening underwent OIT involving sequential up-dosing with 12-hour boiled peanut for 12 weeks, 2-hour boiled peanut for 20 weeks and roasted peanut for 20 weeks, to a target maintenance dose of 12 roasted peanuts daily. PRIMARY OUTCOME: proportion of children passing open-label oral food challenge involving cumulative administration of 12 roasted peanuts (12 g peanuts; approximately 3000 mg peanut protein) 6-8 weeks after reaching the target maintenance dose. Secondary outcomes included treatment-related adverse events and use of medications for treating allergy symptoms. RESULTS: Between 1 July 2017 and 22 June 2018, 70 participants were enrolled and commenced OIT. Desensitization was successfully induced in 56 of 70 (80%) participants. Withdrawal due to treatment-related adverse events was infrequent (n = 3). Treatment-related adverse events were reported in 43 (61%) participants, corresponding to a rate of 6.58 per 1000 OIT doses. Medication use associated with treatment-related adverse events was infrequent, with rescue epinephrine use reported by three (4%) participants (0.05 per 1000 doses). CONCLUSION: Oral immunotherapy using boiled followed by roasted peanuts represents a pragmatic approach that appears effective in inducing desensitization and is associated with a favourable safety profile.

Proteomic analysis of nasal mucus samples of healthy patients and patients with chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) has a complex and multifactorial pathogenesis with a heterogeneous inflammatory profile. Proteomic analysis of nasal mucus may enable further understanding of protein abundances and biologic processes present in CRS and its endotypes compared with in healthy patients. OBJECTIVE: Our aim was to determine differences in the nasal mucus proteome of healthy patients and patients with CRS. METHODS: Nasal mucus was obtained from healthy patients, patients with CRS without nasal polyps (CRSsNP), and patients with CRS with nasal polyps (CRSwNP) before surgery. Gel electrophoresis was performed to fractionate the complex protein extracts before mass spectrometry analysis. Gene set enrichment analysis was performed on differentially expressed proteins. RESULTS: A total of 33 patients were included in this study (12 healthy, 10 with CRSsNP, and 11 with CRSwNP). In all, 1142 proteins were identified in mucus samples from healthy patients, 761 in mucus samples from patients with CRSsNP, and 998 in mucus samples from patients with CRSwNP. Dysfunction in immunologic pathways, reduced cellular signaling, and increased cellular metabolism with associated tissue remodeling pathways were present in patients with CRS compared with in healthy patients. CONCLUSION: Significant downregulation of mucosal immunity and antioxidant pathways with increased tissue modeling processes may account for the clinical manifestations of CRS. Ultimately, the differing proteome and biologic processes provide further insight into CRS pathogenesis and its endotypes.

Abundance of Synaptic Vesicle-Related Proteins in Alpha-Synuclein-Containing Protein Inclusions Suggests a Targeted Formation Mechanism.

Proteinaceous α-synuclein-containing inclusions are found in affected brain regions in patients with Parkinson's disease (PD), Dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). These appear in neurons as Lewy bodies in both PD and DLB and as glial cytoplasmic inclusions (GCIs) in oligodendrocytes in MSA. The role they play in the pathology of the diseases is unknown, and relatively little is still known about their composition. By purifying the inclusions from the surrounding tissue and comprehensively analysing their protein composition, vital clues to the formation mechanism and role in the disease process may be found. In this study, Lewy bodies were purified from postmortem brain tissue from DLB cases (n = 2) and GCIs were purified from MSA cases (n = 5) using a recently improved purification method, and the purified inclusions were analysed by mass spectrometry. Twenty-one percent of the proteins found consistently in the GCIs and LBs were synaptic-vesicle related. Identified proteins included those associated with exosomes (CD9), clathrin-mediated endocytosis (clathrin, AP-2 complex, dynamin), retrograde transport (dynein, dynactin, spectrin) and synaptic vesicle fusion (synaptosomal-associated protein 25, vesicle-associated membrane protein 2, syntaxin-1). This suggests that the misfolded or excess α-synuclein may be targeted to inclusions via vesicle-mediated transport, which also explains the presence of the neuronal protein α-synuclein within GCIs.

Novel protein constituents of pathological ocular pseudoexfoliation syndrome deposits identified with mass spectrometry.

Purpose: Pseudoexfoliation (PEX) syndrome is an age-related progressive disease of the extracellular matrix with ocular manifestations. PEX is clinically diagnosed by the presence of extracellular exfoliative deposits on the anterior surface of the ocular lens. PEX syndrome is a major risk factor for developing glaucoma, the leading cause of irreversible blindness in the world, and is often associated with the development of cataract. PEX reportedly coexists with Alzheimer disease and increases the risk of heart disease and stroke. PEX material deposited on the anterior surface of the ocular lens is highly proteinaceous, complex, and insoluble, making deciphering the protein composition of the material challenging. Thus, to date, only a small proportion of the protein composition of PEX material is known. The aim of this study was to decipher the protein composition of pathological PEX material deposited on the ocular lens in patients and advance the understanding of pathophysiology of PEX syndrome. Methods: Liquid-chromatography and tandem mass spectrometry (LC-MS/MS) was employed to discover novel proteins in extracts of neat PEX material surgically isolated from patients (n = 4) with PEX syndrome undergoing cataract surgery. A sub-set of the identified proteins was validated with immunohistochemistry using lens capsule specimens from independent patients (n=3); lens capsules from patients with cataract but without PEX syndrome were used as controls (n=4). Expression of transcripts of the validated proteins in the human lens epithelium was analyzed with reverse transcription PCR (RT-PCR). Functional relationships among the proteins identified in this study and genes and proteins previously implicated in the disease were bioinformatically determined using InnateDB. Results: Peptides corresponding to 66 proteins, including ten proteins previously known to be present in PEX material, were identified. Thirteen newly identified proteins were chosen for validation. Of those proteins, 12 were found to be genuine components of the material. The novel protein constituents include apolipoproteins (APOA1 and APOA4), stress response proteins (CRYAA and PRDX2), and blood-related proteins (fibrinogen and hemoglobin subunits), including iron-free hemoglobin. The gene expression data suggest that the identified stress-response proteins and hemoglobin are contributed by the lens epithelium and apolipoproteins and fibrinogen by the aqueous humor to the PEX material. Pathway analysis of the identified novel protein constituents and genes or proteins previously implicated in the disease reiterated the involvement of extracellular matrix organization and degradation, elastic fiber formation, and complement cascade in PEX syndrome. Network analysis suggested a central role of fibronectin in the pathophysiology of the disease. The identified novel protein constituents of PEX material also shed light on the molecular basis of the association of PEX syndrome with heart disease, stroke, and Alzheimer disease. Conclusions: This study expands the understanding of the protein composition of pathological PEX material deposited on the ocular lens in patients with PEX syndrome and provides useful insights into the pathophysiology of this disease. This study together with the previous study by our group (Sharma et al. Experimental Eye Research 2009;89(4):479-85) demonstrate that using neat PEX material, devoid of the underlying lens capsule, for proteomics analysis is an effective approach for deciphering the protein composition of complex and highly insoluble extracellular pathological ocular deposits present in patients with PEX syndrome.

Proteomic analysis reveals downregulation of housekeeping proteins in the diabetic vascular proteome.

AIMS: Type 2 diabetes (T2D) increases the risk of death associated with cardiovascular complications. However, a complete understanding of protein changes within the diabetic vasculature is still lacking. METHODS: Herein, we utilized mass spectrometry to perform vascular and urinary proteome analysis using a rat model of high-fat feeding and low-dose streptozotocin to simulate late-stage T2D. The purpose of this study was to identify aortic and urine proteins that are differentially expressed in normal and T2D rats. RESULTS: High-fat feeding and low-dose streptozotocin resulted in hyperglycemia, hypoinsulinemia and high levels of circulating free fatty acids. Using a shotgun proteomic approach, high-mobility-group protein B1 and spondin-1 were significantly increased in T2D aorta compared to control aorta, suggesting vascular inflammation and smooth muscle proliferation, respectively. However, the majority of differentially expressed aortic proteins were downregulated in T2D, including proteins associated with coagulation, cell differentiation and redox homeostasis. Strikingly, we report a significant downregulation of commonly used cytoskeletal housekeeping proteins in T2D aorta. Urine from T2D rats displayed increased expression of proteins involved in inflammation and oxidative stress and decreased expression of proteins associated with lipid metabolism and cell adhesion. A number of differentially expressed proteins in urine of T2D rats have previously been reported in human T2D, thereby supporting this animal model as a good representation of human T2D. CONCLUSIONS: Our data offer new information regarding key pathways that could be therapeutically targeted to combat the cardiovascular complications of T2D.