Serum levels of 4-hydroxynonenal adducts and responding autoantibodies correlate with the pathogenesis from hyperglycemia to Alzheimer's disease.

OBJECTIVE: Hyperglycemia leads to lipid peroxidation, producing 4-hydroxynonenal (HNE) adducts which correlate with the production of amyloid-beta (Aß), one of the hallmarks of Alzheimer's disease (AD). This study is to investigate the interactions of Aß, HNE adducts and responding autoantibodies during the pathogenesis from hyperglycemia to AD. METHODS: A total of 239 Taiwanese serum samples from a healthy control group and patients with hyperglycemia, and AD with and without hyperglycemia were analyzed. Aß was immunoprecipitated from randomly pooled serum in each group and immunoblotted. Synthetic Aß1-16 and Aß17-28 peptides were modified with HNE in vitro and verified with LC-MS/MS. The levels of Aß, HNE adducts, and autoantibody isotypes IgG and IgM against either native or HNE-modified Aß were determined with ELISA. The diagnostic power of potential biomarkers was evaluated. RESULTS: Increased fasting glucose and decreased high-density-lipoprotein cholesterol in AD groups indicated abnormal metabolism in the pathogenesis progression from hyperglycemia to AD. Indeed, serum Aß, HNE adducts and most of the autoantibodies recognizing either native or HNE-modified Aß were increased in the diseased groups. However, HNE adducts had better diagnostic performances than Aß for both hyperglycemia and AD. Additionally, HNE-Aß peptide levels were increased, and the responding autoantibodies (most notably IgM) were decreased in hyperglycemic AD group compared to the hyperglycemia only group, suggesting an immunity disturbance in the pathogenesis progression from hyperglycemia to AD. CONCLUSION: Hyperglycemia increases the level of HNE adducts which may be neutralized by responding autoantibodies. Depletion of these autoantibodies promotes AD-like pathogenesis. Thus, levels of a patient's HNE adducts and associated responding autoantibodies are potential biomarkers for AD with diabetes.

The cathelicidin CATH-2 efficiently neutralizes LPS- and E. coli-induced activation of porcine bone marrow derived macrophages.

Infectious diseases in pigs cause monetary loss to farmers and pose a zoonotic risk. Therefore, it is important to obtain more porcine specific immunological knowledge as a measure to protect against infectious diseases, for example by exploring immunomodulators that are usable as vaccine adjuvants. Cathelicidins are a class of host defence peptides (HDPs) able to directly kill microbes as well as exert a diverse range of effects on the immune system. The peptides have shown promise as immunomodulatory peptides in many applications, including vaccines. However, it is currently unknown what the precise effect of these peptides is on porcine immune cells and whether peptides of other species might also have a strong immunomodulatory effect on porcine macrophages. Mononuclear bone marrow cells of pigs, aged 5-6 months, were cultured into M1 or M2 macrophages and stimulated with LPS or whole bacteria in the presence of host defence peptides (HDPs). CATH-2 and LL-37 strongly inhibited LPS-induced activation of M1 macrophages, the inhibition of LPS-induced activation of M2 macrophages by HDPs was milder, showing that the peptides have selective effects on different cell types. Upon stimulation with whole bacteria, only CATH-2 could effectively inhibit macrophage activation, showing the potent anti-inflammatory potential of this peptide. These results show that porcine peptides are not necessarily the most active in a porcine system, and that CATH-2 is effective in a porcine system as an anti-inflammatory immune modulator, which can be used, for example, in inactivated pathogen vaccines.

Circulating Exosome Involves in the Pathogenesis of Autoimmune Thyroid Diseases Through Immunomodulatory Proteins.

Autoimmune thyroid diseases (AITDs) are chronic organ-specific autoimmune diseases, mainly including Graves' disease (GD) and Hashimoto's thyroiditis (HT). Exosomes, as extracellular vesicles, contain a variety of biologically active substances that play a role in information exchange, thereby affecting the occurrence and progression of diseases. However, it is unclear whether exosomes are involved in the pathogenesis of AITDs. In this study, the role of exosomes in AITDs was explored from a proteomics perspective. Plasma exosomes were isolated from 12 patients with GD, 10 patients with HT, and seven normal controls (NC). Protein profiles were detected using the data-independent acquisition (DIA) method and analyzed to investigate changes in plasma exosome proteins. In the setting of GD, 11 proteins were upregulated while 197 proteins were downregulated compared with healthy people. Among them, MAP1S (log2 FC = 4.669, p = 0.009) and VAMP8 (log2 FC = 3.216, p = 0.003) were the most significantly upregulated, and RSU1 (log2 FC = -6.797, p = 0.001), ACTB (log2 FC = -4.795, p < 0.001), and CXCL7 (log2 FC = -4.674, p < 0.001) were the most significantly downregulated. In the cases of HT, HGFL (log2 FC = 2.766, p = 0.001), FAK1 (log2 FC = 2.213, p < 0.001), and PTN12 (log2 FC = 1.624, p < 0.001) were significantly upregulated, while PSMF1 (log2 FC = -3.591, p < 0.001), PXL2B (log2 FC = -2.622, p = 0.001), and CYTM (log2 FC = -1.609, p < 0.001) were the most downregulated. These differential proteins were mainly enriched in the immune system and metabolic system, indicating that plasma exosomes may play an important role in systemic immune imbalance in AITDs.

Complement Factor H-Related 3 Enhanced Inflammation and Complement Activation in Human RPE Cells.

Complement Factor H-Related 3 (FHR-3) is a major regulator of the complement system, which is associated with different diseases, such as age-related macular degeneration (AMD). However, the non-canonical local, cellular functions of FHR-3 remained poorly understood. Here, we report that FHR-3 bound to oxidative stress epitopes and competed with FH for interaction. Furthermore, FHR-3 was internalized by viable RPE cells and modulated time-dependently complement component (C3, FB) and receptor (C3aR, CR3) expression of human RPE cells. Independently of any external blood-derived proteins, complement activation products were detected. Anaphylatoxin C3a was visualized in treated cells and showed a translocation from the cytoplasm to the cell membrane after FHR-3 exposure. Subsequently, FHR-3 induced a RPE cell dependent pro-inflammatory microenvironment. Inflammasome NLRP3 activation and pro-inflammatory cytokine secretion of IL-1ß, IL-18, IL-6 and TNF-α were induced after FHR-3-RPE interaction. Our previously published monoclonal anti-FHR-3 antibody, which was chimerized to reduce immunogenicity, RETC-2-ximab, ameliorated the effect of FHR-3 on ARPE-19 cells. Our studies suggest FHR-3 as an exogenous trigger molecule for the RPE cell "complosome" and as a putative target for a therapeutic approach for associated degenerative diseases.

Whole Blood Transcriptome Analysis Reveals the Correlation between Specific Immune Cells and Septicemic Melioidosis.

Melioidosis is a serious infectious disease caused by the environmental Gram-negative bacillus Burkholderia pseudomallei. It has been shown that the host immune system, mainly comprising various types of immune cells, fights against the disease. The present study was to specify correlation between septicemic melioidosis and the levels of multiple immune cells. First, the genes with differential expression patterns between patients with septicemic melioidosis (B. pseudomallei) and health donors (control/healthy) were identified. These genes being related to cytokine binding, cell adhesion molecule binding, and MHC relevant proteins may influence immune response. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed 23 enriched immune response pathways. We further leveraged the microarray data to investigate the relationship between immune response and septicemic melioidosis, using the CIBERSORT analysis. Comparison of the percentages of 22 immune cell types in B. pseudomallei vs. control/healthy revealed that those of CD4 memory resting cells, CD8+ T cells, B memory cells, and CD4 memory activated cells were low, whereas those of M0 macrophages, neutrophils, and gamma delta T cells were high. The multivariate logistic regression analysis further revealed that CD8+ T cells, M0 macrophages, neutrophils, and naive CD4+ cells were strongly associated with the onset of septicemic melioidosis, and M2 macrophages and neutrophils were associated with the survival in septicemic melioidosis. Taken together, these data point to a complex role of immune cells on the development and progression of melioidosis.

Establishment of heparin-binding protein time-resolved immunoassay and some potential clinical applications.

AIM: To establish a highly sensitive time-resolved fluorescence immunoassay of heparin-binding protein (HBP-TRFIA) and evaluate its application value for bacterial or fungal infections in tumor patients. METHODS: Two types of HBP monoclonal specific antibodies against different epitopes of the antigen molecule were used as coating antibodies and Eu3+-labeled antibodies, respectively. The double-antibody sandwich method was used in establishing HBP-TRFIA, and the methodology was evaluated. The established HBP-TRFIA was used in detecting HBP concentration in the plasma samples of healthy individuals, patients with bacterial or fungal infections, and infected or uninfected patients with various types of tumors. RESULTS: The linear range of HBP-TRFIA was (0.11-530 ng/mL). Plasma HBP concentrations detected through HBP-TRFIA were consistent with the results of fluorescence quantitative immunochromatography (ρ = 0.964). The plasma HBP concentrations of infected tumor patients were significantly higher than those of uninfected tumor patients (P < 0.01). CONCLUSION: This study successfully established a highly sensitive HBP-TRFIA, which was highly comparable to commercially available fluorescent quantitative immunochromatographic kits and was able to facilitate the timely diagnosis of bacterial or fungal infections in patients with tumor.

The Immunological Factors Predisposing to Severe Covid-19 Are Already Present in Healthy Elderly and Men.

Male sex and old age are risk factors for COVID-19 severity, but the underlying causes are unknown. A possible explanation for this might be the differences in immunological profiles in males and the elderly before the infection. With this in mind, we analyzed the abundance of circulating proteins and immune populations associated with severe COVID-19 in 2 healthy cohorts. Besides, given the seasonal profile of COVID-19, the seasonal response against SARS-CoV-2 could also be different in the elderly and males. Therefore, PBMCs of female, male, young, and old subjects in different seasons of the year were stimulated with heat-inactivated SARS-CoV-2 to investigate the season-dependent anti-SARS-CoV-2 immune response. We found that several T cell subsets, which are known to be depleted in severe COVID-19 patients, were intrinsically less abundant in men and older individuals. Plasma proteins increasing with disease severity, including HGF, IL-8, and MCP-1, were more abundant in the elderly and males. Upon in vitro SARS-CoV-2 stimulation, the elderly produced significantly more IL-1RA and had a dysregulated IFNγ response with lower production in the fall compared with young individuals. Our results suggest that the immune characteristics of severe COVID-19, described by a differential abundance of immune cells and circulating inflammatory proteins, are intrinsically present in healthy men and the elderly. This might explain the susceptibility of men and the elderly to SARS-CoV-2 infection.

Application of Capillary Electrophoresis in Monoclonal Gammopathies and the Cutoff Value of Monoclonal Protein in Differential Diagnosis of Multiple Myeloma and Other Monoclonal Gammopathies.

OBJECTIVE: Monoclonal protein (MP) exists in various diseases, and capillary electrophoresis (CE) has been widely used to detect MP. However, there is not much research on the application value of MP in the differential diagnosis of monoclonal gammopathies. This study aimed to explore MP's cutoff value for the differential diagnosis of multiple myeloma (MM) and other monoclonal gammopathies (MGs). METHODS: A retrospective analysis of 8167 cases was conducted. Serum MP was detected by CE, and the patients' clinical information was collected from the clinical database of our hospital. RESULTS: 985 cases had MP with high peaks, and 91.1% were diagnosed with malignant diseases. The MP showed small peaks in 471 cases, and only 24.4% were diagnosed with malignant diseases. Among the MPs, the IgG-κ type was the most common type, followed by the IgG-λ, IgA-κ, IgA-λ, free λ light chain, IgM-κ, free κ light chain, double clone, and IgM-λ types. Differences in the MP of the IgG, IgA, IgM, and FLC types between the MM group and MGUS group were statistically different (P<0.01). The MP of the IgG, IgA, and FLC types showed clear specificity and sensitivity in discriminating MM from other monoclonal gammopathies in ROC curve analysis. Serum IgM had statistical significance in the differential diagnosis between WM and other MGs (P<0.01). However, there was no statistical significance in the differential diagnosis between MM and other MGs (P=0.140). The cutoff values of the MP of the IgG, IgA, and FLC types were >18.67g/L, >13.86g/L, and >10.15g/L, respectively, for the differential diagnosis of MM and other MGs. The cutoff value of the MP of IgM for the WM diagnosis was >37.75 g/L. CONCLUSION: CE has good clinical application value in the diagnosis of monoclonal gammopathies, and MP can be used in the differential diagnosis of MM and other monoclonal gammopathies.

Bactericidal/Permeability-Increasing Protein Preeminently Mediates Clearance of Pseudomonas aeruginosa In Vivo via CD18-Dependent Phagocytosis.

Chronic Pseudomonas aeruginosa infection mysteriously occurs in the airways of patients with cystic fibrosis (CF), bronchiectasis (BE), and chronic obstructive pulmonary disease (COPD) in the absence of neutrophil dysfunction or neutropenia and is strongly associated with autoimmunity to bactericidal permeability-increasing protein (BPI). Here, we define a critical role for BPI in in vivo immunity against P. aeruginosa. Wild type and BPI-deficient (Bpi-/-) mice were infected with P. aeruginosa, and bacterial clearance, cell infiltrates, cytokine production, and in vivo phagocytosis were quantified. Bpi-/- mice exhibited a decreased ability to clear P. aeruginosa in vivo in concert with increased neutrophil counts and cytokine release. Bpi-/- neutrophils displayed decreased phagocytosis that was corrected by exogenous BPI in vitro. Exogenous BPI also enhanced clearance of P. aeruginosa in Bpi-/- mice in vivo by increasing P. aeruginosa uptake by neutrophils in a CD18-dependent manner. These data indicate that BPI plays an essential role in innate immunity against P. aeruginosa through its opsonic activity and suggest that perturbations in BPI levels or function may contribute to chronic lung infection with P. aeruginosa.

Enhancing clinical and immunological effects of anti-PD-1 with belapectin, a galectin-3 inhibitor.

BACKGROUND: PD-1/PD-L1 engagement and overexpression of galectin-3 (Gal-3) are critical mechanisms of tumor-induced immune suppression that contribute to immunotherapy resistance. We hypothesized that Gal-3 blockade with belapectin (GR-MD-02) plus anti-PD-1 (pembrolizumab) would enhance tumor response in patients with metastatic melanoma (MM) and head and neck squamous cell carcinoma (HNSCC). METHODS: We performed a phase I dose escalation study of belapectin+pembrolizumab in patients with advanced MM or HNSCC (NCT02575404). Belapectin was administered at 2, 4, or 8 mg/kg IV 60 min before pembrolizumab (200 mg IV every 3 weeks for five cycles). Responding patients continued pembrolizumab monotherapy for up to 17 cycles. Main eligibility requirements were a functional Eastern Cooperative Oncology Group status of 0-2, measurable or assessable disease, and no active autoimmune disease. Prior T-cell checkpoint antibody therapy was permitted. RESULTS: Objective response was observed in 50% of MM (7/14) and and 33% of HNSCC (2/6) patients. Belapectin+pembrolizumab was associated with fewer immune-mediated adverse events than anticipated with pembrolizumab monotherapy. There were no dose-limiting toxicities for belapectin within the dose range investigated. Significantly increased effector memory T-cell activation and reduced monocytic myeloid-derived suppressor cells (M-MDSCs) were observed in responders compared with non-responders. Increased baseline expression of Gal-3+ tumor cells and PD-1+CD8+ T cells in the periphery correlated with response as did higher serum trough levels of pembrolizumab. CONCLUSIONS: Belapectin+pembrolizumab therapy has activity in MM and HNSCC. Increased Gal-3 expression, expansion of effector memory T cells, and decreased M-MDSCs correlated with clinical response. Further investigation is planned.

Targeting galectin-3 with a high-affinity antibody for inhibition of high-grade serous ovarian cancer and other MUC16/CA-125-expressing malignancies.

The lectin, galectin-3 (Gal3), has been implicated in a variety of inflammatory and oncogenic processes, including tumor growth, invasion, and metastasis. The interactions of Gal3 and MUC16 represent a potential targetable pathway for the treatment of MUC16-expressing malignancies. We found that the silencing of Gal3 in MUC16-expressing breast and ovarian cancer cells in vitro inhibited tumor cell invasion and led to attenuated tumor growth in murine models. We therefore developed an inhibitory murine monoclonal anti-Gal3 carbohydrate-binding domain antibody, 14D11, which bound human and mouse Gal3 but did not bind human Galectins-1, -7, -8 or -9. Competition studies and a docking model suggest that the 14D11 antibody competes with lactose for the carbohydrate binding pocket of Gal3. In MUC16-expressing cancer cells, 14D11 treatment blocked AKT and ERK1/2 phosphorylation, and led to inhibition of cancer cell Matrigel invasion. Finally, in experimental animal tumor models, 14D11 treatment led to prolongation of overall survival in animals bearing flank tumors, and retarded lung specific metastatic growth by MUC16 expressing breast cancer cells. Our results provide evidence that antibody based Gal3 blockade may be a viable therapeutic strategy in patients with MUC16-expressing tumors, supporting further development of human blocking antibodies against Gal3 as potential cancer therapeutics.

Metabolic mediators determine the association of antinuclear antibody subtypes with specific clinical symptoms in systemic sclerosis.

PURPOSE: The aim of this study was to investigate the possible link between different types of systemic sclerosis-specific antinuclear antibodies, adipokines and endothelial molecules which were recently found to have a pathogenic significance in systemic sclerosis. MATERIALS/METHODS: Serum concentration of adiponectin, resistin, leptin, endothelin-1, fractalkine and galectin-3 were determined in the sera of patients with systemic sclerosis (n â€‹= â€‹100) and healthy controls (n â€‹= â€‹20) using ELISA. RESULTS: The following associations between antinuclear antibodies and increased serum concentrations were identified: anticentromere antibodies with endothelin-1 (p â€‹< â€‹0.0001; mean level in patients 2.21 vs control group 1.31 â€‹pg/ml), anti-topoisomerase I antibodies with fractalkine (p â€‹< â€‹0.0001; 3.68 vs 1.68 â€‹ng/ml) and galectin-3 (p â€‹= â€‹0.0010, 6.39 vs 3.26 â€‹ng/ml). Anti-RNA polymerase III antibodies were associated with increased resistin (p â€‹< â€‹0.0001; 15.13 vs 8.54 â€‹ng/ml) and decreased adiponectin (p â€‹< â€‹0.0001; 2894 vs 8847 â€‹ng/ml). CONCLUSION: In systemic sclerosis metabolic and vascular factors may serve as mediators between immunological abnormalities and non-immune driven clinical symptoms.

Exploration of Serum Marker Proteins in Mice Induced by Babesia microti Infection Using a Quantitative Proteomic Approach.

Babesia microti is a protozoan that mainly parasitizes rodent and human erythrocytes. B. microti infection can result in changes in the expression levels of various proteins in the host serum. To explore the mechanism underlying the regulation of serum proteins by the host during B. microti infection, this study used a data-independent acquisition (DIA) quantitative proteomic approach to perform comprehensive quantitative proteomic analysis on the serum of B. microti-infected mice. We identified and analysed 333 serum proteins during the infectious stage and recovery stage within 30 days of infection by B. microti in mice. Through quantitative analysis, we found 57 proteins differentially expressed in the infection stage and 69 proteins differentially expressed in the recovery stage. Bioinformatics analysis revealed that these differentially expressed proteins were mainly concentrated in organelles, cell parts, and extracellular regions that are mainly involved in immune system, metabolic, and cellular processes. Additionally, the differentially expressed proteins mainly had catalytic activity. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis showed that many of the differentially expressed proteins participate in the complement and coagulation cascade reaction, including complement C3, complement FP, and coagulation factor XII. The results of this study can provide more information for the selection of biomarkers for the early clinical monitoring of babesiosis and help in the treatment of babesiosis.

Human serum proteins bind to Sporothrix schenckii conidia with differential effects on phagocytosis.

Serum is an important source of proteins that interact with pathogens. Once bound to the cell surface, serum proteins can stimulate the innate immune system. The phagocytosis of Sporothrix schenckii conidia by human macrophages is activated through human serum opsonisation. In this study, we have attempted to characterise human blood serum proteins that bind to the cell wall of S. schenckii conidia. We systematically observed the same four proteins independent of the plasma donor: albumin, serum amyloid protein (SAP), α-1 antitrypsin (AAT), and transferrin were identified with the help of tandem mass spectrometry. Phagocytosis depended on the concentration of the SAP or α-1 antitrypsin that was used to opsonise the conidia; however, transferrin or albumin did not have any effect on conidia internalisation. The presence of mannose did not affect macrophage phagocytosis of the conidia opsonised with SAP or α-1 antitrypsin, which suggests that these proteins are not recognised by the mannose receptor.

Helicobacter pylori lipopolysaccharide structural domains and their recognition by immune proteins revealed with carbohydrate microarrays.

The structural diversity of the lipopolysaccharides (LPSs) from Helicobacter pylori poses a challenge to establish accurate and strain-specific structure-function relationships in interactions with the host. Here, LPS structural domains from five clinical isolates were obtained and compared with the reference strain 26695. This was achieved combining information from structural analysis (GC-MS and ESI-MSn) with binding data after interrogation of a LPS-derived carbohydrate microarray with sequence-specific proteins. All LPSs expressed Lewisx/y and N-acetyllactosamine determinants. Ribans were also detected in LPSs from all clinical isolates, allowing their distinction from the 26695 LPS. There was evidence for 1,3-d-galactans and blood group H-type 2 sequences in two of the clinical isolates, the latter not yet described for H. pylori LPS. Furthermore, carbohydrate microarray analyses showed a strain-associated LPS recognition by the immune lectins DC-SIGN and galectin-3 and revealed distinctive LPS binding patterns by IgG antibodies in the serum from H. pylori-infected patients.

Epithelial Cell-Associated Galectin-3 Activates Human Dendritic Cell Subtypes for Pro-Inflammatory Cytokines.

There is mounting evidence that galectin-3 is a prognostic and diagnostic biomarker associated with diverse diseases and conditions, including cancer, cardiovascular disease, autoimmunity, wound healing, allergic disease, and chronic inflammation in general. Yet, whether and exactly how galectin-3 may participate in the pathogenesis of these diseases remains poorly understood. Recently, we have linked the expression of galectin-3 on the A549 epithelial cell line -an adenocarcinoma, to the activation of human basophils for the release of histamine and secretion of IL-4 and IL-13. These responses proved dependent on cell-to-cell contact, basophil expression of IgE, were inhibited by n-acetyllactosamine, and were ablated when basophils were co-cultured with A549 clones lacking galectin-3 expression. While recombinant galectin-3 failed to activate basophils when in solution, microspheres expressing this lectin did so by mimicking the responses seen when using A549 cells. Given the IgE dependency of the basophil responses, and the fact that galectin-3 is long known to bind this immunoglobulin, we hypothesize that a similar mode of activation extends to other IgE-bearing cells. To investigate this possibility, we tested epithelial cell-associated galectin-3 for its capacity to activate human dendritic cells, including the plasmacytoid and myeloid subtypes as well as monocytes, all of which bind IgE. Indeed, results indicate that epithelial cell-associated galectin-3 activated these cells for robust production of TNF-α and IL-6 and up-regulated the expression of activation markers found on dendritic cells. Moreover, many of the same parameters previously observed for basophils applied to the findings herein, including evidence that matrix-bound galectin-3 (whether on epithelial cells or microspheres) facilitates this mode of activation. In contrast, IgE expression was dispensable for these galectin-3-dependent cytokine responses, implying that this lectin activates dendritic cells (and monocytes) by binding to a glycoprotein other than this immunoglobulin. Overall, these findings further demonstrate how galectin-3 mediates immune cell activation, providing novel insight into how this lectin may promote chronic inflammation underlying the pathogenesis of many diseases.

Plasma Proteome Responses in Salmonid Fish Following Immunization.

Vaccination plays a critical role in the protection of humans and other animals from infectious diseases. However, the same vaccine often confers different protection levels among individuals due to variation in genetics and/or immunological histories. While this represents a well-recognized issue in humans, it has received little attention in fish. Here we address this knowledge gap in a proteomic study of rainbow trout (Oncorhynchus mykiss, Walbaum), using non-lethal repeated blood sampling to establish the plasma protein response of individual fish following immunization. Six trout were immunized with adjuvanted hen egg-white lysozyme (HEL) and peripheral blood sampled at ten time points from day 0 to day 84 post-injection. We confirm that an antigen-specific antibody response to HEL was raised, showing differences in timing and magnitude among individuals. Using label-free liquid chromatography-mass spectrometry, we quantified the abundance of 278 plasma proteins across the timecourse. As part of the analysis, we show that this approach can distinguish many (but not all) duplicated plasma proteins encoded by paralogous genes retained from the salmonid-specific whole genome duplication event. Global variation in the plasma proteome was predominantly explained by individual differences among fish. However, sampling day explained a major component of variation in abundance for a statistically defined subset of 41 proteins, representing 15% of those detected. These proteins clustered into five groups showing distinct temporal responses to HEL immunization at the population level, and include classical immune (e.g. complement system members) and acute phase molecules (e.g. apolipoproteins, haptoglobins), several enzymes and other proteins supporting the immune response, in addition to evolutionarily conserved molecules that are as yet uncharacterized. Overall, this study improves our understanding of the fish plasma proteome, provides valuable marker proteins for different phases of the immune response, and has implications for vaccine development and the design of immune challenge experiments.