Identification of novel biomarkers of acute phase response in chickens challenged with Escherichia coli lipopolysaccharide endotoxin.

BACKGROUND: The chicken's inflammatory response is an essential part of the bird's response to infection. A single dose of Escherichia coli (E. coli) lipopolysaccharide (LPS) endotoxin can activate the acute phase response (APR) and lead to the production of acute phase proteins (APPs). In this study, the responses of established chicken APPs, Serum amyloid A (SAA) and Alpha-1-acid-glycoprotein (AGP), were compared to two novel APPs, Hemopexin (Hpx) and Extracellular fatty acid binding protein (Ex-FABP), in 15-day old broilers over a time course of 48 h post E.coli LPS challenge. We aimed to investigate and validate their role as biomarkers of an APR. Novel plant extracts, Citrus (CTS) and cucumber (CMB), were used as dietary supplements to investigate their ability to reduce the inflammatory response initiated by the endotoxin. RESULTS: A significant increase of established (SAA, AGP) and novel (Ex-FABP, Hpx) APPs was detected post E.coli LPS challenge. Extracellular fatty acid binding protein (Ex-FABP) showed a similar early response to SAA post LPS challenge by increasing ~ 20-fold at 12 h post challenge (P < 0.001). Hemopexin (Hpx) showed a later response by increasing ∼5-fold at 24 h post challenge (P < 0.001) with a similar trend to AGP. No differences in APP responses were identified between diets (CTS and CMB) using any of the established or novel biomarkers. CONCLUSIONS: Hpx and Ex-FABP were confirmed as potential biomarkers of APR in broilers when using an E. coli LPS model along with SAA and AGP. However, no clear advantage for using either of dietary supplements to modulate the APR was identified at the dosage used.

Plasma proteomic profiling of bacterial cold water disease-resistant and -susceptible rainbow trout lines and biomarker discovery.

Genetic variation for disease resistance is present in salmonid fish; however, the molecular basis is poorly understood, and biomarkers of disease susceptibility/resistance are unavailable. Previously, we selected a line of rainbow trout for high survival following standardized challenge with Flavobacterium psychrophilum (Fp), the causative agent of bacterial cold water disease. The resistant line (ARS-Fp-R) exhibits over 60 percentage points higher survival compared to a reference susceptible line (ARS-Fp-S). To gain insight into the differential host response between genetic lines, we compared the plasma proteomes from day 6 after intramuscular challenge. Pooled plasma from unhandled, PBS-injected, and Fp-injected groups were simultaneously analyzed using a TMT 6-plex label, and the relative abundance of 513 proteins was determined. Data are available via ProteomeXchange, with identifier PXD041308, and the relative protein abundance values were compared to mRNA measured from a prior, whole-body RNA-seq dataset. Our results identified a subset of differentially abundant intracellular proteins was identified, including troponin and myosin, which were not transcriptionally regulated, suggesting that these proteins were released into plasma following pathogen-induced tissue damage. A separate subset of high-abundance, secreted proteins were transcriptionally regulated in infected fish. The highest differentially expressed protein was a C1q family member (designated complement C1q-like protein 3; C1q-LP3) that was upregulated over 20-fold in the infected susceptible line while only modestly upregulated, 1.8-fold, in the infected resistant line. Validation of biomarkers was performed using immunoassays and C1q-LP3, skeletal muscle troponin C, cathelcidin 2, haptoglobin, leptin, and growth and differentiation factor 15 exhibited elevated concentration in susceptible line plasma. Complement factor H-like 1 exhibited higher abundance in the resistant line compared to the susceptible line in both control and challenged fish and thus was a baseline differentiator between lines. C1q-LP3 and STNC were elevated in Atlantic salmon plasma following experimental challenge with Fp. In summary, these findings further the understanding of the differential host response to Fp and identifies salmonid biomarkers that may have use for genetic line evaluation and on-farm health monitoring.

Faecal proteomics in the identification of biomarkers to differentiate canine chronic enteropathies.

Canine chronic enteropathy (CCE) is a collective term used to describe a group of idiopathic enteropathies of dogs that result in a variety of clinical manifestations of intestinal dysfunction. Clinical stratification into food-responsive enteropathy (FRE) or non-food responsive chronic inflammatory enteropathy (CIE), is made retrospectively based on response to treatments. Faecal extracts from those with a FRE (n = 5) and those with non-food responsive chronic inflammatory enteropathies (CIE) (n = 6) were compared to a healthy control group (n = 14) by applying TMT-based quantitative proteomic approach. Many of the proteins with significant differential abundance between groups were pancreatic or intestinal enzymes with pancreatitis-associated protein (identified as REG3α) and pancreatic M14 metallocarboxypeptidase proteins carboxypeptidase A1 and B identified as being of significantly increased abundance in the CCE group. The reactome analysis revealed the recycling of bile acids and salts and their metabolism to be present in the FRE group, suggesting a possible dysbiotic aetiology. Several acute phase proteins were significantly more abundant in the CCE group with the significant increase in haptoglobin in the CIE group especially notable. Further research of these proteins is needed to fully assess their clinical utility as faecal biomarkers for differentiating CCE cases. SIGNIFICANCE: The identification and characterisation of biomarkers that differentiate FRE from other forms of CIE would prove invaluable in streamlining clinical decision-making and would avoid costly and invasive investigations and delays in implementing effective treatment. Many of the proteins described here, as canine faecal proteins for the first time, have been highlighted in previous human and murine inflammatory bowl disease (IBD) studies initiating a new chapter in canine faecal biomarker research, where early and non-invasive biomarkers for early clinical stratification of CCE cases are needed. Pancreatitis-associated protein, pancreatic M14 metallocarboxypeptidase along with carboxypeptidase A1 and B are identified as being of significantly increased abundance in the CCE groups. Several acute phase proteins, were significantly more abundant in the CCE group notably haptoglobin in dogs with inflammatory enteropathy. The recognition of altered bile acid metabolism in the reactome analysis in the FRE group is significant in CCE which is a complex condition incorporating of immunological, dysbiotic and faecal bile acid dysmetabolism. Both proteomics and immunoassays will enable the characterisation of faecal APPs as well as other inflammatory and immune mediators, and the utilisation of assays, validated for use in analysis of faeces of veterinary species will enable clinical utilisation of faecal matrix to be fully realised.

Milk and serum proteomes in subclinical and clinical mastitis in Simmental cows.

Bovine mastitis causes changes in the milk and serum proteomes. Here changes in both proteomes caused by naturally occurring subclinical and clinical mastitis have been characterised and quantified. Milk and serum samples from healthy dairy cows (n = 10) were compared to those of cows with subclinical (n = 12) and clinical mastitis (n = 10) using tandem mass tag (TMT) proteomics. Proteins that significantly increased or decreased in milk (n = 237) or serum (n = 117) were quantified and classified by the type of change in subclinical and clinical mastitis. A group of the proteins (n = 38) showed changes in both milk and serum a number of which decreased in the serum but increased in milk, suggesting a particular role in host defence for maintaining and restoring homeostasis during the disease. Proteins affected by bovine mastitis included proteins in host defence and coagulation pathways. Investigation of the modified proteomes in milk and serum was assessed by assays for haptoglobin, serum amyloid A and α1 acid glycoprotein validating the results obtained by quantitative proteomics. Alteration of abundance patterns of milk and serum proteins, together with pathway analysis reveal multiple interactions related to proteins affected by mastitis. Data are available via ProteomeXchange with identifier PXD022595. SIGNIFICANCE: Mastitis is the most serious condition to affect dairy cows and leads to reduced animal welfare as well as having a negative economic effect for the dairy industry. Proteomics has previously identified changes in abundance of milk proteins during mastitis, but there have been few investigations addressing changes that may affect proteins in the blood during the infection. In this study, changes in the abundance of proteins of milk and serum, caused by naturally occurring mastitis have been characterised by proteomics using a quantitative approach and both subclinical and clinical cases of mastitis have been investigated. In both milk and serum, change in individual proteins was determined and classified into varying types of altering abundance, such as increasing in subclinical mastitis, but showing no further increase in clinical mastitis. Of special interest were the proteins that altered in abundance in both milk and serum which either showed similar trends - increasing or decreasing in both biological fluids or showed reciprocal change decreasing in serum but increasing in milk. As well as characterising proteins as potential markers of mastitis and the severity of the disease, these results provide insight into the pathophysiology of the host response to bovine mastitis.

Global production capacity of seasonal and pandemic influenza vaccines in 2019.

Vaccines will be an important element in mitigating the impact of an influenza pandemic. While research towards developing universal influenza vaccines is ongoing, the current strategy for vaccine supply in a pandemic relies on seasonal influenza vaccine production to be switched over to pandemic vaccines. Understanding how much vaccine could be produced, in which regions of the world and in what timeframe is critical to informing influenza pandemic preparedness. Through the Global Action Plan for Influenza Vaccines, 2006-2016, WHO promoted an increase in vaccine production capacity and monitors the landscape through periodically surveying influenza vaccine manufacturers. This study compares global capacity for production of influenza vaccines in 2019 with estimates from previous surveys; provides an overview of countries with established production facilities; presents vaccine production by type and manufacturing process; and discusses limitations to these estimates. Results of the current survey show that estimated annual seasonal influenza vaccine production capacity changed little since 2015 increasing from 1.47 billion to 1.48 billion doses with potential maximum annual influenza pandemic vaccine production capacity increasing from 6.37 billion to 8.31 billion doses. However, this figure should be interpreted with caution as it presents a best-case scenario with several assumptions which may impact supply. Further, pandemic vaccines would not be immediately available and could take four to six months for first supplies with several more months needed to reach maximum capacity. A moderate-case scenario is also presented of 4.15 billion doses of pandemic vaccine in 12 months. It is important to note that two doses of pandemic vaccine are likely to be required to elicit an adequate immune response. Continued efforts are needed to ensure the sustainability of this production and to conduct research for vaccines that are faster to produce and more broadly protective taking into account lessons learned from COVID-19 vaccine development.

Quantitative proteomics of cerebrospinal fluid using tandem mass tags in dogs with recurrent epileptic seizures.

This prospective study included four dog groups (group A: healthy dogs, groups B: dogs with idiopathic epilepsy under antiepileptic medication (AEM), C: idiopathic epilepsy dogs without AEM administration, D: dogs with structural epilepsy). The purpose of the study was to compare the proteomic profile among the four groups. Samples were analyzed by a quantitative Tandem Mass Tags approach using a Q-Exactive-Plus mass-spectrometer. Identification and relative quantification were performed using Proteome Discoverer, and data were analyzed using R. Gene ontology terms were analyzed based on Canis lupus familiaris database. Data are available via ProteomeXchange with identifier PXD018893. Eighteen proteins were statistically significant among the four groups (P < 0.05). MMP2 and EFEMP2 appeared down-regulated whereas HP and APO-A1 were up-regulated (groups B, D). CLEC3B and PEBP4 were up-regulated whereas APO-A1 was down-regulated (group C). IGLL1 was down-regulated (groups B, C) and up-regulated (group D). EFEMP2 was the only protein detected among the four groups and PEBP4 was significantly different among the epileptic dogs. Western blot and SPARCL immunoassay were used to quantify HP abundance change, validating proteomic analysis. Both, showed good correlation with HP levels identified through proteomic analysis (r = 0.712 and r = 0.703, respectively). SIGNIFICANCE: The proteomic analysis from CSF of dogs with epileptic seizures could reflect that MMP2, HP and APO-A1 may contribute to a blood-brain barrier disruption through the seizure-induced inflammatory process in the brain. MMP2 change may indicate the activation of protective mechanisms within the brain tissue. Antiepileptic medication could influence several cellular responses and alter the CSF proteome composition.

Concentrations of Acute-Phase Proteins in Milk from Cows with Clinical Mastitis Caused by Different Pathogens.

Among the new diagnostic methods for mastitis detection under development, milk acute-phase proteins (APPs) are receiving special attention. The study aimed to compare the profile of milk APPs from cows with natural clinical mastitis caused by distinct pathogens. The concentrations of haptoglobin (Hp), serum amyloid A (SAA), alpha-1-acid glycoprotein (AGP), and C-reactive protein (CRP) were measured by Spatial Proximity Analyte Reagent Capture Luminescence (SPARCL). Each APP was compared across the pathogens causing mastitis. The APPs differed statistically (p < 0.05) among the pathogens causing udder infection. There were significant and positive correlations among the concentration profile, for each pathogen, in three of four APPs studied. It can be concluded that the pathogen causing mastitis could modify the profile of release of the APPs in milk. The profile of Hp, AGP, and CRP demonstrated significant correlation, indicating that the three APPs are suggested as biomarkers, in milk, for bovine mastitis.

Cardiovascular-renal axis disorder and acute-phase proteins in cats with congestive heart failure caused by primary cardiomyopathy.

BACKGROUND: Currently, the pathogenesis of congestive heart failure (CHF) in cats is not fully understood. OBJECTIVE: To identify novel biomarkers for CHF in cats caused by primary cardiomyopathy, particularly related to cardiovascular-renal axis disorder and systemic inflammatory response. ANIMALS: Twenty-five cats in CHF caused by primary cardiomyopathy, 12 cats with preclinical cardiomyopathy, and 20 healthy controls. METHODS: Case control and observational case series. The following serum biomarkers were compared among the 3 cat groups: a cardiorenal profile that included N-terminal pro-brain natriuretic peptide (NT-proBNP), symmetric dimethylarginine (SDMA), and creatinine and an inflammatory profile that included 7 acute-phase proteins (APPs). Survival analyses and longitudinal studies were performed in CHF cats. RESULTS: All cardiorenal biomarkers were positively correlated and higher in CHF cats, and high NT-proBNP and SDMA were associated with poor clinical outcome. Cats with CHF had significantly higher leucine-rich alpha-2-glycoprotein 1, serum amyloid A, and ceruloplasmin, and these APPs were positively correlated with NT-proBNP and left atrial size. In a multivariable survival analysis, alpha-1-acid glycoprotein concentration (P = .01), body weight (P = .02) and left atrial-to-aortic root ratio (P = .01) were independent prognostic factors for CHF in these cats. CONCLUSIONS AND CLINICAL IMPORTANCE: In cats, CHF is an inflammatory disorder and outcome in CHF may be determined by the extent of inflammation and possibly the amount of residual renal function. These novel biomarkers have potential use for the clinical management, prognosis, and future research into CHF and cardiomyopathy in cats.

The plasma proteome and the acute phase protein response in canine pyometra.

Canine pyometra is a common inflammatory disease of uterus in sexually mature bitches caused by secondary bacterial infection, leading to change in plasma proteins associated with the innate immune system. Proteomic investigation is increasingly being applied to canine diseases in order to identify and quantify significant changes in the plasma proteome. The aim of the study was to assess and quantify changes in plasma proteome profiles of healthy dogs and pyometra affected bitches using a TMT-based high-resolution quantitative proteomic approach. As a result, 22 proteins were significantly down-regulated including transthyretin, antithrombin, retinol-binding protein, vitamin D binding protein, paraoxonase 1, and kallikrein, while 16 were significantly up-regulated including haptoglobin light chain, alpha-1-acid glycoprotein, C-reactive protein precursor, and lipopolysaccharide-binding protein in dogs with pyometra. Pathway analysis indicated that acute inflammatory response, regulation of body fluid levels, protein activation cascade, the humoral immune response, and phagocytosis were affected in pyometra. Validation of biological relevance of the proteomic study was evident with significant increases in the concentrations of haptoglobin, C-reactive protein, alpha-1-acid glycoprotein, and ceruloplasmin by immunoassay. Pyometra in bitches was shown to stimulate an increase in host defence system proteins in response to inflammatory disease including the acute phase proteins. SIGNIFICANCE: The label-based high-resolution quantitative proteomics analysis and bioinformatic approach used in this study provide insight into the complex pathophysiology of inflammation associated with pyometra revealing proteins with biomarker potential. Early diagnosis and therapeutic intervention may prevent severe complications associated with advancing sepsis in dogs with pyometra. Therefore the identification of diagnostic biomarkers that, after clinical validation may be used in veterinary practice and protein relevant to pathways responding to disease are important findings of the study. Data are available via ProteomeXchange with identifier PXD015951.

Considerations for sustainable influenza vaccine production in developing countries.

Through its Global Action Plan for Influenza Vaccines (GAP), the World Health Organization (WHO) in collaboration with the United States Department of Health and Human Services has produced a checklist to support policy-makers and influenza vaccine manufacturers in identifying key technological, political, financial, and logistical issues affecting the sustainability of influenza vaccine production. This checklist highlights actions in five key areas that are beneficial for establishing successful local vaccine manufacturing. These five areas comprise: (1) the policy environment and health-care systems; (2) surveillance systems and influenza evidence; (3) product development and manufacturing; (4) product approval and regulation; and (5) communication to support influenza vaccination. Incorporating the checklist into national vaccine production programmes has identified the policy gaps and next steps for countries involved in GAP's Technology Transfer Initiative. Lessons learnt from country experiences provide context and insight that complement the checklist's goal of simplifying the complexities of influenza prevention, preparedness, and vaccine manufacturing.

Estrogen receptor dependent inhibitors of NF-kappaB transcriptional activation-1 synthesis and biological evaluation of substituted 2-cyanopropanoic acid derivatives: pathway selective inhibitors of NF-kappaB, a potential treatment for rheumatoid arthritis.

Pathway selective ligands of the estrogen receptor inhibit transcriptional activation of proinflammatory genes mediated by NF-kappaB. Substituted 2-cyanopropanoic acid derivatives were developed leading to the discovery of WAY-204688, an orally active, pathway selective, estrogen receptor dependent anti-inflammatory agent. This propanamide was shown to be orally active in preclinical models of inflammatory diseases, such as rheumatoid arthritis, without the proliferative effect associated with traditional estrogens.

Control of chronic inflammation with pathway selective estrogen receptor ligands.

The discovery of novel intervention points in the inflammatory pathway has been a focus of drug development in recent years. We have identified pathway selective ligands for the estrogen receptor (ER) that inhibit NF-kappaB mediated inflammatory gene expression causing a reduction of cytokines, chemokines, adhesion molecules and inflammatory enzymes. SAR development of a series of 4-(Indazol-3-yl)-phenols has led to the identification of WAY-169916 an orally active non-steroidal ligand with the potential use in the treatment of inflammatory diseases without the classical proliferative effects associated with non-selective estrogens.

Synthesis and activity of a new class of pathway-selective estrogen receptor ligands: hydroxybenzoyl-3,4-dihydroquinoxalin-2(1H)-ones.

The anti-inflammatory activity of non-selective estrogens has been attributed to their ability to antagonize the activity of nuclear factor kappaB (NF-kappaB), a known mediator of inflammatory responses. Here we report the identification of a potent new class of pathway-selective ER ligands that selectively antagonize NF-kappaB functional activity, while exhibiting a lack of classical estrogenic effect.

Substituted 4-hydroxyphenyl sulfonamides as pathway-selective estrogen receptor ligands.

The transcription factor nuclear factor-kappaB (NF-kappaB) is a key component in the onset of inflammation. We describe here a series of 4-hydroxyphenyl sulfonamide estrogen receptor (ER) ligands that selectively inhibit NK-kappaB transcriptional activity but are devoid of conventional estrogenic activity.

The activity of pathway-selective estrogen receptor ligands in experimental septic shock.

Estrogen receptors (ER) are widely expressed in multiple genital and nongenital tissues. Upon engagement of these receptors, multiple genes are affected in target tissues via estrogen response elements. Nonsteroidal pathway-selective ER ligands have recently been identified that inhibit NF-kappaB transcriptional activity and are devoid of conventional estrogenic activities on genital tissues. These pathway-selective ligands are potent anti-inflammatory agents in vivo and may prove to be of therapeutic utility in systemic inflammatory states. These pathway-selective ER ligands were tested in the murine listeriosis model, the neutropenic rat model, and the mouse cecal ligation and puncture model. WAY-204688 did not have any significant activity after systemic infection by Listeria monocytogenes. In the neutropenic rat model, WAY-204688 provided a significant survival benefit against an otherwise lethal challenge of Pseudomonas aeruginosa 12.4.4 compared with the control group (88% versus 25% survival; P < 0.05). Preservation of mucosal weight and prevention of histopathologic changes were observed with the administration of WAY-204688. Similar findings were observed in a cecal ligation and puncture model with WAY-204688 and a related compound WAY-169916. These results indicate that oral administration of these pathway-selective ER ligands preserved gastrointestinal barrier function and improve outcome in experimental models of systemic infection and inflammation. These agents may prove to be useful clinically as a novel treatment strategy for severe sepsis.