Expression Of Acute Phase Proteins Research Articles

Prolonged hyperinsulinemia increases the production of inflammatory cytokines in equine digital lamellae but not in striated muscle

Hyperinsulinemia is the key feature of equine metabolic syndrome (EMS) which leads to debilitating sequelae. Hyperinsulinemia-associated laminitis (HAL) is one of the major sequelae of EMS, although the pathophysiological mechanisms are not well elucidated. Using an equine model, we hypothesized that expression of inflammatory markers would be increased in digital lamellae and striated muscle following prolonged hyperinsulinemia. Healthy Standardbred horses (5.4 ± 1.9 years) were alternately assigned to a prolonged euglycemic-hyperinsulinemic clamp (pEHC) or control group (n = 4 per group). Following a 48 h pEHC or a 48 h infusion of a balanced electrolyte solution (controls), biopsies were collected from digital lamellar tissue, skeletal muscle and cardiac muscle were obtained. All hyperinsulinemic horses developed laminitis regardless of previous health status at enrollment. Protein expression was quantified via Western blotting. A significant (P < 0.05) upregulation of the protein expression of heat shock protein 90 (HSP90), alpha 2 macroglobulin (A2M) and fibrinogen (α, β isoforms), as well as inflammatory cytokines including interleukin-1β were detected in digital lamellae following prolonged hyperinsulinemia. In contrast, protein expression of cytokines and acute phase proteins in heart and skeletal muscle was unchanged following hyperinsulinemia. Upregulation of inflammatory cytokines and acute phase proteins in digital lamellae during prolonged hyperinsulinemia may reveal potential biomarkers and novel therapeutic targets for equine endocrinopathic laminitis. Further, the lack of increase of inflammatory proteins and acute phase proteins in striated muscle following prolonged hyperinsulinemia may highlight potential anti-inflammatory and cardioprotective mechanisms in these insulin-sensitive tissues.

Cell-Specific Regulation of Inflammatory Cytokines and Acute-Phase Proteins by the Glucocorticoid Receptor

Background. Literature and data mining found abnormal induction of chemokine (C-X-C motif) ligand 1 (CXCL1) and CXCL8 and down-regulation of CXCL2 in inflammatory liver diseases. This study was performed to understand the glucocorticoid receptor’s (GR’s) effects on chemokine and acute-phase protein expression in human liver, in settings of bacterial infection (modeled using LPS) or inflammation (modeled using TNFα). Methods. Primary human hepatocytes (PHH) were treated with combinations of tumor necrosis factor alpha (TNFα), lipopolysaccharide (LPS), and dexamethasone (DEX) for 24 h, following which chemokine mRNA and protein expression were analyzed using qPCR and enzyme-linked immunosorbent assay assays. Dual luciferase assays were performed on transfected cell lines. Mutant CXCL2 promoters were used in dual luciferase assays to identify specific regions of the CXCL2 promoter affected by GR, TNFα, or hepatocyte nuclear factor 4α (HNF4α, a liver-enriched transcription factor). Results. In PHH from donor 1, GR strongly inhibited LPS-induced CXCL1 and CXCL8 translation and transcription, whereas CXCL2 transcription tended to increase with DEX treatment. In PHH from donor 2, DEX treatment inhibited protein expression and secretion of CXCL1 and CXCL8 induced by TNFα and/or LPS, whereas CXCL2 upregulation was largely unaffected by DEX treatment. In nonliver HEK293T cells GR activity inhibited CXCL2 promoter activity. However, in liver-derived HEPG2 cells, GR induced CXCL2 promoter activity. A 407-base pair region upstream of CXCL2 promoter is necessary for full GR functionality in HEPG2 cells. TNFα synergized with HNF4α in inducing CXCL2 promoter activity in HEPG2 cells. Conclusions. GR’s effects on chemokine expression are cell-type specific and chemokine specific. GR down-regulated CXCL1 and CXCL8 in different cell types, whereas the specific activation of CXCL2 in hepatocytes and down-regulation of CXCL2 in nonhepatocytes by GR appears due to cell-specific utilization of CXCL2 promoter. By specifically increasing GR activity in the liver, we may normalize chemokine imbalances and prevent sepsis in inflammatory liver diseases.

Serum alpha 1-acid glycoprotein and interleukin-8 elevations in felines with localized and metastatic tumors.

This study aimed to investigate the expression of acute phase proteins and plasma cytokines in cats with various tumor types and varying metastatic statuses. 5 clinically healthy cats and 22 cats with neoplastic disease that underwent CT imaging before treatment were enrolled. Patients were grouped based on their tumor types and metastatic status. Blood samples were collected from all cats for general blood analyses before they underwent CT imaging. The remaining plasma sample was frozen for subsequent alpha 1-acid glycoprotein (AGP), serum amyloid A (SAA), and feline cytokine panel measurements. These results were compared with those of healthy cats as well as between metastatic status and tumor types. Only 4 cats (18%) exhibited elevated SAA levels, whereas 16 (73%) showed elevated AGP levels. AGP was significantly increased in cats with tumors (P = .016), while SAA was not. Only IL-8 showed a significant difference (P = .002) between cats with primary tumors and those with radiologically suspected tumor metastasis. While AGP is a more prominent biomarker than SAA in cats with tumors, a significant elevation of AGP and SAA levels in association with metastasis and specific tumor types could not be identified. Alternatively, further investigation is warranted to evaluate the potential significance of IL-8 in tumor progression and metastasis.

Vitamin D status affects proteomic profile of HDL-associated proteins and inflammatory mediators in dyslipidemia

Vitamin D deficiency and dyslipidemia have substantial implications for human health globally. Vitamin D is essential for bone metabolism and immune modulation, and its insufficiency is linked to various chronic inflammatory conditions. Dyslipidemia, characterized by low levels of high-density lipoprotein (HDL) and elevated levels of low-density lipoprotein (LDL) and triglycerides, is also prevalent. Previous research has shown a connection between vitamin D deficiency and low HDL, but the precise mechanism by which vitamin D influences HDL production and its anti-inflammatory properties remains unclear. This study aimed to investigate the proteomic profiles of individuals with and without vitamin D deficiency and dyslipidemia, specifically focusing on the effects of vitamin D on HDL production, its anti-inflammatory potential, and the molecular pathways associated with vitamin D deficiency and dyslipidemia, particularly inflammation and cancer pathways. By analyzing the proteomic profiles of 274 participants from the Qatar Biobank database, we identified 1301 proteins. Our findings indicated a decrease in HDL-associated apolipoproteins (ApoM and ApoD) in individuals with both dyslipidemia and vitamin D deficiency. Conversely, participants with these conditions exhibited increased expression of acute-phase proteins (SAA1 and SOD1), which are associated with inflammation. Pathway enrichment analysis revealed heightened inflammatory activity in individuals with vitamin D deficiency and dyslipidemia, with notable enrichments in pathways such as MAPK, JAK-STAT, Ras signaling, cytokine-cytokine receptor interaction, AGE-RAGE, ErbB signaling, and cancer pathways. Overall, cases of vitamin D deficiency showed enrichment in inflammation pathways, while individuals with both vitamin D deficiency and dyslipidemia demonstrated enhanced activation of cancer and inflammation pathways.

Influence of the concentrate inclusion level in a grass silage–based diet on hepatic transcriptomic profiles in Holstein-Friesian dairy cows in early lactation

Excessive negative energy balance in early lactation is linked to an increased disease risk but may be mitigated by appropriate nutrition. The liver plays central roles in both metabolism and immunity. Hepatic transcriptomic profiles were compared between 3 dietary groups in each of 40 multiparous and 18 primiparous Holstein-Friesian cows offered isonitrogenous grass silage-based diets with different proportions of concentrates: (1) low concentrate (LC, 30% concentrate + 70% grass silage); (2) medium concentrate (MC, 50% concentrate + 50% grass silage), or (3) high concentrate (HC, 70% concentrate + 30% grass silage). Liver biopsies were taken from all cows at around 14 d in milk for RNA sequencing, and blood metabolites were measured. The sequencing data were analyzed separately for primiparous and multiparous cows using CLC Genomics Workbench V21 (Qiagen Digital Insights), focusing on comparisons between HC and LC groups. More differentially expressed genes (DEG) were seen between the primiparous cows receiving HC versus LC diets than for multiparous cows (597 vs. 497), with only 73 in common, indicating differential dietary responses. Multiparous cows receiving the HC diet had significantly higher circulating glucose and insulin-like growth factor-1 and lower urea than those receiving the LC diet. In response to HC, only the multiparous cows produced more milk. In these animals, bioinformatic analysis indicated expression changes in genes regulating fatty acid metabolism and biosynthesis (e.g., ACACA, ELOVL6, FADS2), increased cholesterol biosynthesis (e.g., CYP7A1, FDPS, HMGCR), downregulation in hepatic AA synthesis (e.g., GPT, GCLC, PSPH, SHMT2), and decreased expression of acute phase proteins (e.g., HP, LBP, SAA2). The primiparous cows on the HC diet also downregulated genes controlling AA metabolism and synthesis (e.g., CTH, GCLC, GOT1, ODC1, SHMT2) but showed higher expression of genes indicative of inflammation (e.g., CCDC80, IL1B, S100A8) and fibrosis (e.g., LOX, LUM, PLOD2). This potentially adverse response to a HC diet in physically immature animals warrants further investigation.

Study of the Genetic Expression of Antiretroviral Restriction Factors and Acute Phase Proteins in Cattle Infected with Bovine Leukemia Virus.

The goal of this study was to analyze the genetic expression of antiretroviral restriction factors (ARF) and acute phase proteins (APP), as well as their correlation with proviral and viral loads in cattle with aleukemic (AL) and persistent lymphocytosis (PL). Complete blood samples were collected from a herd of dairy cows, and we extracted genetic material from peripheral blood leukocytes. Absolute quantification of the expression of ARF (APOBEC-Z1, Z2, and Z3; HEXIM-1, HEXIM-2, and BST2) and APP (haptoglobin (HP), and serum amyloid A (SAA)) was performed by qPCR. Statistical significance was observed in the expression of APOBEC-Z3 in BLV-infected animals. We only found positive correlations with a strong expression of the ARF genes in the AL group. The participation of APOBEC (Z1 and Z3), HEXIM-1, and HEXIM-2 was more frequently identified in BLV-infected animals. HEXIM-2 showed active gene expression in the AL group. Although the expression of ARF in early stages of infection (AL) maintains an important participation, in late stages (PL) it seems to have little relevance.

Equine insulin dysregulation causes tissue specific alterations of proinflammatory cytokines and acute phase proteins in a NF-kB independent manner

Similar to human diabetes, equine metabolic syndrome (EMS) causes insulin dysregulation leading to debilitating sequela including laminitis. The pathophysiological mechanisms underlying EMS and laminitis are not well known. Therefore, using an insulin-resistant equine model, we hypothesized that insulin dysregulation induces an increased expression of inflammatory proteins in a tissue specific manner. Two groups of horses (n = -5/group) were categorized as insulin-resistant (IR) or insulin-sensitive (IS), using a frequently sampled intra-venous glucose tolerance test. Biopsies from skeletal muscle, and visceral and subcutaneous adipose tissues were collected in both groups. Protein expression was quantified via Western blotting in order to investigate HSP90, α 2 macroglobulin (A2M), Fibrinogen α, β, γ isoforms as well as cytokines, including interleukin-1β (IL-1β) and interleukin-6 (IL-6), in muscle and adipose tissues. Protein expression of HSP90, A2M and IL1-β was significantly greater in visceral adipose tissue of IR horses compared to IS horses. Fibrinogen (α and γ) expression was only significantly increased in subcutaneous adipose tissue of IR group compared to IS group. In contrast, no statistically significant difference in protein expression of proinflammatory cytokines and acute phase proteins was reported in skeletal muscle of IR vs. IS horses. Relative protein expression of total and phospho-NFκB protein expression was not statistically significantly changed in adipose tissues of IR horses compared to IS horses. In conclusion, proinflammatory cytokines and acute phase proteins were upregulated in adipose tissue, but not in skeletal muscle, through an NF-kB independent pathway. Insights from this study could reveal novel biomarkers and potential therapeutic targets for EMS.

A high-concentrate diet provokes inflammatory responses by downregulating Forkhead box protein A2 (FOXA2) through epigenetic modifications in the liver of dairy cows

A high-concentrate diet has been reported to promote an inflammatory response in dairy cows. The purpose of this study was to clarify the effect of the high-concentrate (HC) diet on hepatic Forkhead box protein A2 (FOXA2) expression and uncover the molecular mechanisms in inflammatory responses in the liver. The results showed that the HC diet reduced the ruminal fluid pH and elevated the secretion of SAA3, IL-1α, and IL-8 and reduced that of IL-10 in peripheral blood plasma. Compared with the low-concentrate (LC) group, the concentration of myeloperoxidase (MPO) was higher in the liver of dairy cows in the HC group. In addition, the relative mRNA expression of acute phase proteins (HP, SAA3, and LBP), proinflammatory cytokines (TNFα, IL-1α, IL-1β, IL-8), TLR4, MyD88, TRAF6, TRIF, IκBα, p65, p38 and JNK1 was upregulated and that of IL-10 was downregulated in the liver of the HC group. Consistently, the protein abundance of TLR4, TNFα and phosphorylation of proteins involved in NF-κB (IκBα and p65) and MAPK (p38 and JNK) pathways were significantly increased in the HC group compared with the LC group. And both the mRNA and protein abundance of FOXA2 were downregulated in the HC group. Further epigenetic analysis results demonstrated that chromatin compaction and DNA hypermethylation contributed to inhibiting FOXA2 expression, in which the demethylase ten-eleven translocation 1 (TET1) and histone deacetylase 3 (HDAC3) might participate. Overall, these findings demonstrated that the high-concentrate diet triggered inflammatory cascades and downregulated FOXA2 by epigenetic modifications in the liver of dairy cows.

Tregs Attenuate Peripheral Oxidative Stress and Acute Phase Proteins in ALS.

Oxidative stress (OS) induces inflammation, which in turn exacerbates OS and the expression of acute phase proteins (APPs). Regulatory T lymphocyte (Treg) therapy was assessed for suppression of OS and APP responses in longitudinal serum samples from subjects with amyotrophic lateral sclerosis (ALS) enrolled in a phase I clinical trial. The first round of Treg therapy suppressed levels of oxidized low‐density lipoprotein (ox‐LDL). During a 6‐month washout period, ox‐LDL levels increased. A second round of therapy again suppressed ox‐LDL levels and then rose following the cessation of treatment. Serum levels of APPs, soluble CD14, lipopolysaccharide binding protein, and C‐reactive protein, were stabilized during Treg administrations, but rose during the washout period and again after therapy was discontinued. Treg therapy potentially suppresses peripheral OS and the accompanying circulating pro‐inflammatory induced APPs, both of which may serve as peripheral candidates for monitoring efficacies of immunomodulating therapies. ANN NEUROL 2022;92:195–200

Profiling the Expression of Circulating Acute-Phase Proteins, Cytokines, and Checkpoint Proteins in Patients with Severe Trauma: A Pilot Study.

PurposeSevere trauma may lead to the systemic release of inflammatory mediators into the circulation with profound acute-phase responses; however, the understanding of the expression of these mediators remains limited. This study aimed to characterize the alterations in the expression of circulating acute-phase proteins, cytokines, and checkpoint proteins in patients with severe trauma injuries.Patients and MethodsThe study population included trauma patients in the intensive care unit (ICU) with an injury severity score equal to or greater than 16 and who had used a ventilator for 48 hours. A total of 12 female and 28 male patients were recruited for the study; six patients died and 34 survived. Blood samples collected at acute stages were compared with those drawn at the subacute stage, the time when the patients were discharged from the ICU, or before the discharge of the patients from the hospital.ResultsThe study identified that the expression of acute-phase proteins, such as alpha-1-acid glycoprotein and C-reactive protein, and cytokines, including granulocyte colony-stimulating factor, interleukin-6, and interleukin-1 receptor antagonist, was elevated in the circulation after severe trauma. In contrast, the levels of acute-phase proteins, such as alpha-2-macroglobulin, serum amyloid P, and von Willebrand factor, and cytokines, including interleukin-4 and interferon gamma-induced protein 10, were reduced. However, there were no significant differences in the expression of checkpoint proteins in the circulation.ConclusionThe dysregulated proteins identified in this study may serve as potential therapeutic targets or biomarkers for treating patients with severe trauma. However, the related biological functions of these dysregulated factors require further investigation to validate their functions.

Acute-phase protein synthesis: a key feature of innate immune functions of the liver.

The expression of acute-phase proteins (APP's) maintains homeostasis and tissue repair, but also represents a central component of the organism's defense strategy, especially in the context of innate immunity. Accordingly, an inflammatory response is accompanied by significant changes in the serum protein composition, an aspect that is also used diagnostically. As the main site of APP synthesis the liver is constantly exposed to antigens or pathogens via blood flow, but also to systemic inflammatory signals originating either from the splanchnic area or from the circulation. Under both homeostatic and acute-phase response (APR) conditions the composition of APP's is determined by the pattern of regulatory mediators derived from the systemic circulation or from local cell populations, especially liver macrophages. The key regulators mentioned here most frequently are IL-1β, IL-6 and TNF-α. In addition to a variety of molecular mediators described mainly on the basis of in vitro studies, recent data emphasize the in vivo relevance of cellular key effectors as well as molecular key mediators and protein modifications for the regulation and function of APP's. These are aspects, on which the present review is primarily focused.

Plasma Exosome Profile in ST-Elevation Myocardial Infarction Patients with and without Out-of-Hospital Cardiac Arrest.

The identification of new biomarkers allowing an early and more accurate characterization of patients with ST-segment elevation myocardial infarction (STEMI) is still needed, and exosomes represent an attractive diagnostic tool in this context. However, the characterization of their protein cargo in relation to cardiovascular clinical manifestation is still lacking. To this end, 35 STEMI patients (17 experiencing resuscitated out-of-hospital cardiac arrest (OHCA-STEMI) and 18 uncomplicated) and 32 patients with chronic coronary syndrome (CCS) were enrolled. Plasma exosomes were characterized by the nanoparticle tracking analysis and Western blotting. Exosomes from STEMI patients displayed a higher concentration and size and a greater expression of platelet (GPIIb) and vascular endothelial (VE-cadherin) markers, but a similar amount of cardiac troponin compared to CCS. In addition, a difference in exosome expression of acute-phase proteins (ceruloplasmin, transthyretin and fibronectin) between STEMI and CCS patients was found. GPIIb and brain-associated marker PLP1 accurately discriminated between OHCA and uncomplicated STEMI. In conclusion, the exosome profile of STEMI patients has peculiar features that differentiate it from that of CCS patients, reflecting the pathophysiological mechanisms involved in STEMI. Additionally, the exosome expression of brain- and platelet-specific markers might allow the identification of patients experiencing ischemic brain injury in STEMI.

Cyclic GMP-AMP promotes the acute phase response and protects against Escherichia coli infection in mice

The acute phase response, as a component of the innate immune system, is part of the first line of defense against invading pathogens. The Stimulator of Interferon Genes (STING) pathway initiates innate immune responses upon recognition of exogenous bacterial and viral DNA. However, whether STING signaling pathway plays any roles in regulating acute phase response during bacterial infection remains unknown. In this study, we used STING-deficient (Tmem173gt) and wildtype mice to investigate acute phase responses to bacterial infection (Escherichia coli, E. coli) and test the effect of exogenous cyclic GMP-AMP (cGAMP, a STING agonist) treatment. Bacterial infection of STING-deficient mice resulted in an increase in mortality and bacterial dissemination. Also, inflammation-induced acute phase response was drastically reduced in STING-deficient mice, showing significant reduction in expression of cytokine TNF-α and acute phase proteins. In contrast, exogenous cGAMP treatment enhanced inflammation-induced acute phase response by increasing the expression of TNF-α and acute phase proteins. Also, cGAMP accelerated bacterial clearance and improved survival rate of wildtype mice, but not STING-deficient mice. Interestingly, cGAMP treatment mitigated bacterial infection induced liver injury in both wildtype and STING-deficient mice. Further in vitro evidence showed that cGAMP treatment retarded TNF-α-mediated hepatocyte apoptosis, potentially accelerating autophagy. Taken together, our results indicated that cGAMP/STING signaling pathway is critical for organism to initiate blood-borne innate immune-responses to defend bacterial infection, and cGAMP is envisaged as a drug candidate for further clinical trial.

アスコルビン酸欠乏は，腸管でのIL-6 産生を上昇させて肝臓のSTAT3 を活性化することにより急性期タンパク質の発現を上昇させる

アスコルビン酸欠乏は，腸管でのIL-6 産生を上昇させて肝臓のSTAT3 を活性化することにより急性期タンパク質の発現を上昇させる

17α-ethinylestradiol and 4-tert-octylphenol concurrently disrupt the immune response of common carp

The aquatic environment is massively polluted with endocrine-disrupting compounds (EDCs) including synthetic estrogens (e.g. 17α-ethinylestradiol, EE2) and alkylphenols (e.g. 4-tert-octylphenol, 4t-OP). A major mechanism of action for estrogenic EDCs is their interaction with estrogen receptors and consequently their modulation of the action of enzymes involved in steroid conversion e.g. aromatase CYP19.We now studied the effects of EE2 and 4t-OP on the anti-bacterial immune response of common carp. We investigated effects on the number/composition of inflammatory leukocytes and on the gene expression of mediators that regulate inflammation and EDC binding. In vitro we found that high concentrations of both EE2 and 4t-OP down-regulated IFN-γ2 and IFN-γ-dependent immune responses in LPS-stimulated monocytes/macrophages. Similarly, during bacterial infection in fish, in vivo treated with EE2 and 4t-OP, decreased gene expression of il-12p35 and of ifn-γ2 was found in the focus of inflammation. Moreover, during A. salmonicida-induced infection in EE2-treated carp, but not in fish fed with 4t-OP-treated food, we found an enhanced inflammatory reaction manifested by high number of inflammatory peritoneal leukocytes, including phagocytes and higher expression of pro-inflammatory mediators (inos, il-1β, cxcl8_l2). Furthermore, in the liver, EE2 down-regulated the expression of acute phase proteins: CRPs and C3. Importantly, both in vitro and in vivo, EDCs altered the expression of estrogen receptors: nuclear (erα and erβ) and membrane (gpr30). EDCs also induced up-regulation of the cyp19b gene.Our findings reveal that contamination of the aquatic milieu with estrogenic EDCs, may considerably violate the subtle and particular allostatic interactions between the immune response and endogenous estrogens and this may have negative consequences for fish health.

Effects of supplemental calcium butyrate on lamb growth, plasma metabolites, and hepatic gluconeogenic and inflammatory gene expression changes

Objective was to evaluate the effects of increasing supplemental calcium butyrate on intake, growth, plasma acute phase proteins and hepatic enzyme expression in lambs fed a concentrate-based diet. Twelve pens of crossbred Hampshire × Suffolk lambs (n = 2 lambs/pen) were assigned to 1 of 3 dietary treatments: (1) no butyrate (C), (2) 6.25 g calcium butyrate/kg of diet DM (B1), and (3) 12.5 g calcium butyrate/kg of diet DM (B2). All lambs were adapted to a basal concentrated-based diet for 2 weeks prior to the start of the experiment. Treatment diets were fed from d 0–56. Blood was collected on d 0, 28, and 55 for analysis of plasma β-hydroxybutyric acid (BHBA), total protein, glucose, ceruloplasmin (CP), and haptoglobin (HP). Intakes were recorded daily by pen and individual body weights were measured weekly. Liver tissue was collected via biopsy on d 56 to measure mRNA expression of gluconeogenic enzymes, acute phase proteins, and inflammatory transcription factors using NanoString. Orthogonal contrasts were used to determine the effects of increasing dietary inclusion of Ca butyrate on lamb growth performance. Interactions of treatment by day were examined for plasma concentrations collected over time. Lambs fed B1 had 67 % and 40 % greater average daily gain (ADG) (quadratic; P = 0.02) than the lambs fed C and B2, respectively. Similarly, lambs fed B1 had 13.4 % and 15.8 % greater DMI (quadratic; P = 0.02) than the lambs fed C and B2, respectively. Total protein, BHBA, glucose, and CP were not affected (P ≥ 0.12) by treatment. There was a treatment × day interaction (P = 0.01) on plasma HP. Plasma HP concentrations did not change in lambs fed B1. Plasma HP concentrations declined on d 55 compared to d 28 in lambs fed C; but, plasma HP concentrations increased on d 55 compared to d 28 in lambs fed B2. In the liver, mRNA expression of acute phase proteins and anti-inflammatory transcription factors were not affected (P ≥ 0.24) by calcium butyrate inclusion in the diet. Similar to DMI and ADG, gluconeogenic enzymes decreased (quadratic; P ≤ 0.08) in lambs fed B1 compared to the lambs fed C and B2. This decrease in mRNA expression of gluconeogenic enzymes may be associated with the feedback from increased DMI in lambs fed B1. Lambs fed B1 achieved greater DMI and ADG when compared to the lambs fed B2 and C and these performance results may be linked to key gluconeogenic pathways.

Ascorbic acid deficiency induces hepatic and intestinal expression of inflammation-related genes irrespective of the presence or absence of gut microbiota in ODS rats

We have previously demonstrated that ascorbic acid (AsA) deficiency causes inflammatory changes in the liver and intestine in Osteogenic Disorder Shionogi (ODS) rats, which are unable to synthesize AsA. We have suggested that AsA deficiency increased intestinal interleukine (IL)-6 production, stimulating hepatic acute phase proteins (APPs) expression via the portal vein. In this study, we determined whether these hepatic and intestinal inflammatory changes by AsA deficiency are induced in germ-free (GF) ODS rats. For 18 days, male specific pathogen-free (SPF) ODS rats were fed the basal diet containing 600 mg AsA/kg (control group) or the AsA-free diet (AsA-deficient group) in SPF conditions, while male GF ODS rats were fed the basal diet (control group) or the AsA-free diet (AsA-deficient group) in GF conditions. Firstly, AsA deficiency significantly elevated the hepatic expression of APPs in both SPF and GF rats. In hepatic mRNA levels of some APPs, significant interaction between GF and AsA-deficiency effects was observed. Secondly, AsA deficiency elevated intestinal IL-6 and IL-1β mRNA levels in both SPF and GF rats, and significant interaction between GF and AsA-deficiency effects was observed in these mRNA levels of jejunum and cecum. In SPF and GF rats, AsA deficiency elevated portal IL-6 concentration. These results show that AsA deficiency caused hepatic and intestinal inflammatory changes in both the GF and SPF ODS rats and indicate that AsA deficiency could directly induce intestinal inflammatory changes without the involvement of gut microbiota.

Inhibition of IRAK4 kinase activity improves ethanol-induced liver injury in mice

Alcohol-related liver disease (ALD) is a major cause of chronic liver disease worldwide with limited therapeutic options. Interleukin-1 receptor associated kinase 4 (IRAK4), the master kinase of Toll-like receptor (TLR)/IL-1R-mediated signalling activation, is considered a novel therapeutic target in inflammatory diseases, but has not been investigated in the context of ALD. IRAK4 phosphorylation and IRAK1 protein were analysed in liver from alcohol-related hepatitis patients and healthy controls. IRAK4 kinase activity-inactive knock-in (Irak4 KI) mice and bone marrow chimeric mice were exposed to chronic ethanol-induced liver injury. IL-1β-induced IRAK4-mediated signalling and acute phase response were investigated in cultured hepatocytes. IRAK1/4 inhibitor was used to test the therapeutic potential for ethanol-induced liver injury in mice. Increased IRAK4 phosphorylation and reduced IRAK1 protein were found in livers of patients with alcoholic hepatitis. In the chronic ethanol-induced liver injury mouse model, hepatic inflammation and hepatocellular damage were attenuated in Irak4 KI mice. IRAK4 kinase activity promotes expression of acute phase proteins in response to ethanol exposure, including C-reactive protein and serum amyloid A1 (SAA1). SAA1 and IL-1β synergistically exacerbate ethanol-induced cell death exvivo. Pharmacological blockage of IRAK4 kinase abrogated ethanol-induced liver injury, inflammation, steatosis, as well as acute phase gene expression and protein production in mice. Our data elucidate the critical role of IRAK4 kinase activity in the pathogenesis of ethanol-induced liver injury in mice and provide preclinical validation for use of an IRAK1/4 inhibitor as a new potential therapeutic strategy for the treatment of ALD. Herein, we have identified the role of IRAK4 kinase activity in the development of alcohol-induced liver injury in mice. Hepatocyte-specific IRAK4 is associated with an acute phase response and release of proinflammatory cytokines/chemokines, which synergistically exacerbate alcohol-induced hepatocyte cell death exvivo. Pharmacological inhibition of IRAK4 kinase activity effectively attenuates alcohol-induced liver injury in mice and could have therapeutic implications.

Evaluation of blood-based proteomic biomarkers associated with response to anti-PD-1 treatment in NSCLC.

e21531 Background: Immune checkpoint inhibitors have remarkably improved the natural history of patients with non-small cell lung cancer (NSCLC), with improved clinical responses and overall survival compared to standard therapy. However, most unselected NSCLC patients do not respond, highlighting the need of theranostic biomarker discovery. In a cohort of NCSLC patients treated with anti-PD-1-blockade, we investigate baseline blood-based parameters with a high-throughput quantitative proteomics workflow, based on data independent acquisition mass spectrometry (DIA-MS). Quantified proteins are evaluated as potential circulating biomarkers for predicting the response to therapy. Methods: Plasma samples from 49 NSCLC subjects treated with anti-PD-1-blockade were prepared for mass spectrometry. All samples were analyzed on a Thermo Scientific Q Exactive HF-X operated in a DIA mode using capillary flow liquid chromatography with separation over a 40 min gradient. DIA data was extracted with Spectronaut to quantify expressed proteins. Results: Across all samples, 605 proteins were quantified and 17 were significantly associated (log2 fold change &gt; 0.58 and q-value &lt; 0.05) with Responders (SD + PR) vs Non-Responder (PD) groups. Functional mapping and unsupervised clustering of regulated proteins identifies three major subject groups based on protein expression profiles: Group A is characterized by high expression of acute phase proteins (e.g. SAA1/2, CRP), Group B has low overall protein expression, and Group C has high expression glycolysis related enzymes (ALDOA, GAPDH) and focal adhesion proteins (VCL). Consistent with previous studies the Group A, with high levels of acute phase proteins, had poor overall survival relative to the other two families (HR = 2.2 [1.2-4.2], p = 0.003). Notably, Group C is comprised largely of Non-Responders (8/12) yet had improved overall survival relative to Group A (HR = 2.5 [1.2-5.0], p = 0.004). Conclusions: Plasma proteomic profiling with capillary flow DIA-MS enables quantification protein profiles in a hypothesis free manner. Poor outcomes on anti-PD-1 therapy was confirmed for subjects with elevated acute phase proteins. However, a subgroup with high focal adhesion and glycolysis proteins had improved survival despite containing non-responder subjects, requiring further investigation.

Acute phase response to exertional heat stroke in mice.

What is the central question of this study? Exertional heat stroke is accompanied by a marked inflammatory response. In this study, we explored the time course of acute phase proteins during recovery from severe heat stress in mice and the potential role of skeletal muscles as their source. What is the main finding and its importance? Exertional heat stroke transiently increased expression of acute phase proteins in mouse liver and plasma and depleted liver and plasma fibrinogen, a typical response to severe trauma. In contrast, skeletal muscle fibrinogen production was stimulated by heat stroke, which can provide an additional reservoir for fibrinogen supply to maintain the clotting potential throughout the body and locally within the muscle. Exertional heat stroke (EHS), the most severe manifestation of heat illness, is accompanied by a marked inflammatory response. The release of acute phase proteins (APPs) is an important component of inflammation, which can assist in tissue survival/repair. The time course of APPs in recovery from EHS is unknown. Furthermore, skeletal muscles produce APPs during infection, but it is unknown whether they can produce APPs after EHS. Our objective was to determine the time course of representative APPs in liver, plasma and skeletal muscle during recovery from EHS. Male C57BL6/J mice ran in a forced running wheel at 37.5°C, 40% relative humidity until symptom limitation. Exercise control (EXC) mice ran for the same duration and intensity at 22.5°C. Samples were collected (n=6-12 per group) over 14days of recovery. Protein abundance was quantified using immunoblots. Total and phosphorylated STAT3 (pSTAT3) at Tyr705, responsible for APP activation, increased in liver at 0.5h after EHS compared with EXC, (P<0.05 and P<0.001, respectively). In contrast, in tibialis anterior (TA) muscle, total STAT3 increased at 3h (P<0.05) but pSTAT3 (Tyr705) did not. Liver serum amyloidA1 (SAA1) increased at 3 and 24h after EHS (P<0.05), whereas plasma SAA1 increased only at 3h (P<0.05). SAA1 was not detected in TA muscle. In liver and plasma, fibrinogen decreased at 3h (P<0.01) and increased in TA muscle (P<0.05). Lipocalin-2 was undetectable in liver or TA muscle. Recovery from EHS is characterized by a transient acute phase response in both liver and skeletal muscle. However, APP expression profiles and subtypes differ between skeletal muscle and liver.