Serum Alpha 2-macroglobulin Research Articles

Salivary expression of novel inflammatory marker Plasminogen Activator Inhibitor-1 in health and disease: an interventional study

Objective: To assess levels of the biomarkers Plasminogen Activator Inhibitor-1 (PAI-1) and alpha 2-macroglobulin (2MG) in saliva and serum in healthy individuals and patients with and without type 2 diabetes mellitus (T2DM) before and after periodontal therapy. Materials and Methods: The 90 recruited subjects were divided into the three following groups: Group 1, with 30 healthy subjects; Group 2, with 30 systemically healthy subjects with stage II and III periodontitis; and Group 3, with 30 patients with periodontitis and well-controlled T2DM (PDM). Salivary and serum PAI-1 and α2MG levels were estimated by enzyme-linked immunosorbent assay and correlated with clinical parameters before and three months after periodontal therapy, and the data obtained was statistically analyzed. Results: There was an improvement in clinical parameters in both groups that underwent initial periodontal therapy. Salivary and serum PAI-1 and α2MG levels were upregulated in PDM group, compared to periodontitis alone at baseline. A significant reduction in the levels of the biomarkers was observed three months after periodontal therapy, and an improvement in glycaemic control was also seen in the PDM group. Conclusion: PAI-1 was expressed in saliva and serum in healthy patients, and increased in periodontitis and diabetes patients, thus evidencing its possible role in periodontal inflammation and glucose regulation, and its levels decreased after periodontal therapy.

Increased serum level of alpha-2 macroglobulin and its production by B-lymphocytes in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL), the most common adult’s leukemia in the western world, is caused in 95% of the cases by uncontrolled proliferation of monoclonal B-lymphocytes. The complement system in CLL is chronically activated at a low level via the classical pathway (CP). This chronic activation is induced by IgG-hexamers, which are formed after binding to alpha-2-macroglobulin (A2M). The study investigated for the first time the serum levels of A2M in CLL patients, their association with the disease severity, and A2M production by the malignant B-lymphocytes. Blood samples were collected from 65 CLL patients and 30 normal controls (NC) subjects, and used for quantifications of the A2M levels, the complement activation marker (sC5b-9), the complement components C2, C3 and C4, and clinical biochemistry and hematology parameters. The production of A2M was studied in B-lymphocytes isolated from blood samples as well as in CLL and non-CLL cell lines.The serum A2M levels were significantly higher in CLL patients vs NCs, showing values of 3.62 ± 0.22 and 1.97 ± 0.10 mg/ml, respectively. Within the CLL group, A2M levels correlated significantly with the disease stage, with sC5b-9, and with clinical indicators of the disease severity. Increased A2M production was showed in three out of four CLL B-lymphocytic lines that were studied, as compared to non-CLL lines, to a non-lymphocytic line, and to blood-derived primary B-lymphocytes. A2M production was further increased both in primary cells and in the CLL cell-line after incubation with CLL sera, compared to NC sera. This study shows for the first time that serum A2M levels in CLL are significantly increased, likely due to A2M production by the malignant B-lymphocytes, and are correlated with the disease severity and with chronic complement activation. The moderate change in A2M production after incubation with NC sera in-vitro supports the hypothesis that inhibition of excess A2M production can be achieved, and that this may potentially down-regulate the IgG-hexamerization and the resulting chronic CP activation. This may also help restore complement system activity, and eventually improve complement activity and immunotherapy outcomes in CLL.

Impact of scaling and root planing on salivary and serum plasminogen activator inhibitor-1 expression in patients with periodontitis with and without type 2 diabetes mellitus.

Plasminogen activator inhibitor-1 (PAI-1) is significantly enhanced in insulin resistance and inflammation and ascribed as a proinflammatory marker. This study aimed to compare and correlate salivary and serum PAI-1 and alpha 2-macroglobulin (α2MG) in patients with periodontitis with and without type 2 diabetes mellitus (T2DM) and also appraise the consequence of periodontal treatment on these biomarkers. Sixty subjects enlisted were split into two groups; Group 1 consisted of 30 systemically healthy subjects with Stage II and III, generalized, Grade B, C periodontitis while Group 2 consisted of 30 patients with periodontitis and well-controlled T2DM (PDM). Salivary and serum PAI-1 and α2MG levels were estimated by enzyme-linked immunosorbent assay and allied with clinical parameters before and 3 months post non-surgical periodontal therapy (NSPT). Data were statistically analyzed using student t-test and Spearman correlation. Analogous improvements in clinical periodontal markers were experienced in both groups after initial periodontal treatment. Estimates of salivary and serum PAI-1 and α2MG were higher among the PDM group compared with periodontitis alone at baseline. Significant diminution in estimates of biomarkers was noted 3 months after NSPT. In the PDM group, there was also an improvement in glycemic control. NSPT positively impacted both groups. Noteworthy expression of salivary and serum PAI-1 in patients with periodontitis and diabetes insinuates a possible role of the adipokine in periodontal inflammation and glucose level regulation. Salivary PAI-1 could thus be used as a diagnostic biomarker to detect disease activity and to track periodontal therapy response.

Clinical Interest of Serum Alpha-2 Macroglobulin, Apolipoprotein A1, and Haptoglobin in Patients with Non-Alcoholic Fatty Liver Disease, with and without Type 2 Diabetes, before or during COVID-19.

In patients with non-alcoholic fatty liver disease (NAFLD) with or without type 2 diabetes mellitus (T2DM), alpha-2 macroglobulin (A2M), apolipoprotein A1 (ApoA1), and haptoglobin are associated with the risk of liver fibrosis, inflammation (NASH), and COVID-19. We assessed if these associations were worsened by T2DM after adjustment by age, sex, obesity, and COVID-19. Three datasets were used: the “Control Population”, which enabled standardization of protein serum levels according to age and sex (N = 27,382); the “NAFLD-Biopsy” cohort for associations with liver features (N = 926); and the USA “NAFLD-Serum” cohort for protein kinetics before and during COVID-19 (N = 421,021). The impact of T2DM was assessed by comparing regression curves adjusted by age, sex, and obesity for the liver features in “NAFLD-Biopsy”, and before and during COVID-19 pandemic peaks in “NAFLD-Serum”. Patients with NAFLD without T2DM, compared with the values of controls, had increased A2M, decreased ApoA1, and increased haptoglobin serum levels. In patients with both NAFLD and T2DM, these significant mean differences were magnified, and even more during the COVID-19 pandemic in comparison with the year 2019 (all p < 0.001), with a maximum ApoA1 decrease of 0.21 g/L in women, and a maximum haptoglobin increase of 0.17 g/L in men. In conclusion, T2DM is associated with abnormal levels of A2M, ApoA1, and haptoglobin independently of NAFLD, age, sex, obesity, and COVID-19.

Trh positive strain of Vibrio parahaemolyticus induce immunity by modulating MAPK pathway: A molecular pathogenic insight in immune-related gene regulation

Vibrio parahaemolyticus is a zoonotic bacterium that causes infections in shellfish, fish and higher vertebrates as well as in humans. The Tdh and Trh positive strains of V. parahaemolyticus are generally considered as major virulent strains. The pathogenic mechanisms of Trh positive strain of V. parahaemolyticus are poorly understood. Therefore, in the present study Indian Major Carp, Labeo rohita was intraperitoneally challenged with a Trh positive strain of V. parahaemolyticus below lethal dose 50 (LD50) to understand the innate immune response. A significant upregulation in the respiratory burst activity, myeloperoxidase activity and lysozyme activity of serum was observed in the challenged fishes. However, the serum alpha (α) 2-macro globulin activity and antiprotease activity remained unaltered in the infected fish. The relative expression study of some immune-related genes showed that after the experimental challenge the expression of immune-related genes viz., Toll-like receptor (TLR), Nucleotide-binding oligomerization domain (NOD), Interleukin-1β (IL-β), Interleukin-6 (IL-6), Tumor necrosis factor α (TNFα), Inducible nitric oxide synthase (iNOS), Complement factor 3a (C3a) and Heat shock proteins 70 (Hsp70) was upregulated during infection. Furthermore, overexpression of nuclear factor kappa light chain enhancer of activated B cells (NF-κβ), Myeloid differentiation primary response 88 (MyD88), Mitogen-activated protein kinases (MAPK) and cysteine-aspartic proteases (Casp 1) was also observed after post-infection which clearly indicated that Trh positive V. parahaemolyticus activates MAPK pathway. The present study strengthens the understanding of molecular pathogenesis and provides insights on gene regulation during infection with Trh positive V. parahaemolyticus.

Quench me if you can: Alpha-2-macroglobulin trypsin complexes enable serum biomarker analysis by MALDI mass spectrometry

One of the main functions of alpha-2-macroglobulin (A2M) in human blood serum is the binding of all classes of protease. It is known that trypsin, after such interaction, possesses modified proteolytic activity. Trypsin first hydrolyzes two bonds in A2M’s ‘bait region’, and the peptide 705VGFYESDVMGR715 is released from A2M. In this work, specifics of the A2M-trypsin interaction were used to determine A2M concentration directly in human blood serum using MALDI mass-spectrometry. Following exogenous addition of trypsin to human blood serum in vitro, the concentration of the VGFYESDVMGR peptide was measured, using its isotopically-labeled analogue (18O), and A2M concentration was calculated. The optimized mass spectrometric approach was verified using a standard method for A2M concentration determination (ELISA) and the relevant statistical analysis methods. It was also shown that trypsin’s modified proteolytic activity in the presence of serum A2M can be used to analyze other serum proteins, including potential biomarkers of pathological processes. Thus, this work describes a promising approach to serum biomarker analysis that can be technically extended in several useful directions.

Alpha-2-Macroglobulin as a New Promising Biomarker Improving the Diagnostic Sensitivity of Bovine Paratuberculosis.

Johne's disease (JD) is a chronic granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP), which induces persistent diarrhea and cachexia. JD causes huge economic losses to the dairy industry due to reduced milk production and premature culling. Infected animals excrete MAP via feces during the prolonged subclinical stage without exhibiting any clinical signs. Therefore, accurate detection of subclinical stage animals is crucial for successful eradication of JD in the herd. In the current study, we analyzed serum samples of MAP-infected and non-infected cattle to identify potential biomarker candidates. First, we identified 12 differentially expressed serum proteins in subclinical and clinical shedder groups compared to the healthy control group. Second, we conducted ELISA for three selected biomarkers (alpha-2-macroglobulin (A2M), alpha-1-beta glycoprotein, and transthyretin) and compared their diagnostic performance with that of two commercial ELISA diagnostic kits. Serum A2M levels were significantly higher in the MAP-exposed, subclinical shedder, subclinical non-shedder, and clinical shedder groups than in the healthy control group, suggesting its possible use as a diagnostic biomarker for MAP infection. Furthermore, A2M demonstrated a sensitivity of 90.4%, and a specificity of 100% while the two commercial ELISA kits demonstrated a sensitivity of 67.83 and 73.04% and a specificity of 100%, respectively. In conclusion, our results suggest that measuring A2M by ELISA can be used as a diagnostic tool to detect MAP infection, considerably improving the detection rate of subclinical shedders and MAP-exposed animals that are undetectable using current diagnostic tools.

Новые белковые маркеры в прогнозе внутриутробной инфекции при преждевременных родах

Objective. To identify prognostic markers of intrauterine infection (IUI) of the fetus and newborn in maternal serum, amniotic fluid, and umbilical cord serum in case of preterm birth. Patients and methods. We examined 93 pregnant women who had preterm birth (PB) from 24 to 33 weeks of gestation. Thirtyfive women delivered babies without any signs of IUI, while 30 women had newborns with mild IUI (conjunctivitis, lymphadenitis, pyoderma) and 28 women had newborns with severe IUI (early neonatal sepsis, advanced herpesvirus infection, chlamydiosis, candidiasis, pneumonia, and meningitis). We measured the levels of alpha 2-macroglobulin (α2-MG) in maternal serum (MS), umbilical cord serum (UCS) using rocket immunoelectrophoresis and in amniotic fluid (AF) using enzyme-linked immunosorbent assay (ELISA). The level of lactoferrin (LF) was assessed using ELISA. MS and UCS levels of albumin (ALB) were measured using biochemical methods, while AF level ALB was evaluated using rocket immunoelectrophoresis. Statistical analysis was performed using logistic regression. Results. We found no association between the concentration of LF in all biological fluids and the condition of newborns. Levels of ALB in MS and UCS also demonstrated no correlation with the condition of newborns. AF ALB in women who delivered babies with IUI (any grade of severity) was significantly higher than that in women who delivered babies without IUI. Women who delivered babies with severe IUI demonstrated the lowest concentration of α2-MG in their serum, whereas women who delivered babies with mild IUI had the highest α2-MG concentration. The AF α2-MG level was 10 to 20 times higher in women who had babies with IUI (any grade of severity) compared to those who had babies without IUI. Conclusion. Low α2-MG level in MS (&lt;2.2 g/L) along with elevated α2-MG level in AF (≥6.0 mg/L) in 86–89% of PB cases indicated generalized fetal IUI and required urgent delivery without prolongation of pregnancy. Key words: albumin, alpha-2-macroglobulin, intrauterine infection, lactoferrin. premature birth

An exploratory study examining how nano-liquid chromatography\u2013mass spectrometry and phosphoproteomics can differentiate patients with advanced fibrosis and higher percentage collagen in non-alcoholic fatty liver disease

BackgroundNon-alcoholic steatohepatitis (NASH) is among the leading causes of liver disease worldwide. It is increasingly recognized that the phenotype of NASH may involve a number of different pathways, of which each could become important therapeutic targets. The aim of this study is to use high resolution mass spectrometry (MS) and phosphoproteomics techniques to assess the serum proteome and hepatic phosphoproteome in subjects with NASH-related fibrosis.MethodsSixty-seven biopsy-proven NAFLD subjects with frozen sera and liver tissue were included. Reverse phase protein microarray was used to quantify the phosphorylation of key signaling proteins in liver and nano-liquid chromatography (LC)-MS was used to sequence target biomarkers in the serum. An image analysis algorithm was used to quantify the percentage of collagen (% collagen) using computer-assisted morphometry. Using multiple regression models, serum proteomes and phosphorylated hepatic proteins that were independently (p ≤ 0.05) associated with advanced fibrosis (stage ≥ 2) and higher % collagen were assessed.ResultsPhosphorylated signaling pathways in the liver revealed that apoptosis signal-regulating kinase 1, mitogen-activated protein kinase (ASK1-MAPK pathway involving ASK1 S38 (p < 0.02) and p38 MAPK (p = 0.0002)) activated by the inflammatory cytokine interleukin (IL-10) (p < 0.001), were independently associated with higher % collagen. LC-MS data revealed that serum alpha-2 macroglobulin (α2M) (p = 0.0004) and coagulation factor V (p = 0.0127) were independently associated with higher % hepatic collagen.ConclusionsSimultaneous profiling of serum proteome and hepatic phosphoproteome reveals that the activation of ASK1 S38, p38 MAPK in the liver, and serum α2M and coagulation factor V are independently associated with hepatic collagen deposition in patients with NASH. These data suggest the role of these pathways in the pathogenesis of NASH-related fibrosis as a potential therapeutic target.

Beneficial effects of natural eggshell membrane (NEM) on multiple indices of arthritis in collagen-induced arthritic rats

Objectives: This study was performed to evaluate the potential efficacy of natural eggshell membrane (NEM) in collagen-induced arthritic rats, a well-established rodent model of inflammation and rheumatoid arthritis.Methods: Rats with developing type II collagen-induced arthritis (CIA) were treated once daily by oral gavage on study days –14 to 17 with vehicle or NEM (52 mg/kg body weight). Rats were euthanized on study day 17. Efficacy was assessed by daily ankle caliper measurements, ankle diameter expressed as area under the curve (AUCd0–17), and histopathologic evaluation of ankles and knees. Serum biomarkers of cartilage function and inflammation [collagen type II C-telopeptide (CTXII), cartilage oligomeric matrix protein (COMP), and alpha-2-macroglobulin (A2M)] were measured by ELISA.Results: Treatment with NEM resulted in significant beneficial effects on the daily ankle diameter measurements and ankle diameter AUC. Ankle and knee histopathology scores were significantly reduced (36% and 43% reduction of summed individual histopathology scores for ankle and knee, respectively; p < 0.05) toward normal for rats given NEM compared to vehicle controls. The percent reduction of serum CTXII, COMP, and A2M in NEM-treated rats ranged from 30% to 72% (p < 0.05).Conclusions: NEM significantly improved multiple aspects of inflammatory arthritis including inflammation, pannus, cartilage damage, bone resorption, and periosteal bone formation. This study provides further support for the use of CTXII, COMP, and A2M as relevant biomarkers that were responsive to NEM.

Alpha-2 macroglobulin in Alzheimer's disease: a marker of neuronal injury through the RCAN1 pathway.

Preclinical changes that precede the onset of symptoms and eventual diagnosis of Alzheimer's disease (AD) are a target for potential preventive interventions. A large body of evidence suggests that inflammation is closely associated with AD pathogenesis and may be a promising target pathway for such interventions. However, little is known about the association between systemic inflammation and preclinical AD pathophysiology. We first examined whether the acute-phase protein, alpha-2 macroglobulin (A2M), a major component of the innate immune system, was associated with cerebrospinal fluid (CSF) markers of neuronal injury in preclinical AD and risk of incident AD in the predictors of cognitive decline among normal individuals (BIOCARD) cohort. We find that A2M concentration in blood is significantly associated with CSF concentrations of the neuronal injury markers, tau and phosphorylated tau, and that higher baseline serum A2M concentration is associated with an almost threefold greater risk of progression to clinical symptoms of AD in men. These findings were replicated in the Alzheimer's Disease Neuroimaging (ADNI) study. Then, utilizing a systems level approach combining large multi-tissue gene expression datasets with mass spectrometry-based proteomic analyses of brain tissue, we identified an A2M gene network that includes regulator of calcineurin (RCAN1), an inhibitor of calcineurin, a well-characterized tau phosphatase. A2M gene and protein expression in the brain were significantly associated with gene and protein expression levels of calcineurin. Collectively these novel findings suggest that A2M is associated with preclinical AD, reflects early neuronal injury in the disease course and may be responsive to tau phosphorylation in the brain through the RCAN1-calcineurin pathway.

118. The interplay between psychological well-being, pain and inflammation in a rat model of spinal cord injury

We hypothesize that inflammation, inherent to spinal cord injury (SCI), contributes to decreased psychological well-being. To test this, male Sprague–Dawley rats ( N = 47) received a mild, moderate, or severe contusion or were intact controls. Behavioral measures of anxiety, depression and pain were collected prior to injury, and on days 10 and 21 post injury. Behavioral tests assessed anhedonia (sucrose preference), anxiety (center activity and shock probe burying), psychomotor activity (open field activity) and social interest (social exploration). Pain tests assessed mechanical allodynia (tactile reactivity) and thermal hyperalgesia (tail-flick). Serum alpha-2 macroglobulin levels were also measured on Days 1, 10 and 21, and thymus weight was recorded on Day 25. First, subjects’ average scores (Days 10 and 21) on behavioral measures of depression and anxiety were subjected to principal components and hierarchical cluster analyses. The analyses derived three subject clusters, identified via ANOVAs as: “Depression/Anxiety” ( n = 15), “Depression” ( n = 12) and “Healthy” ( n = 20). The “Depression/Anxiety” cluster showed increased mechanical allodynia, anxiety, and peripheral inflammation relative to the “Healthy” cluster. The “Depression” cluster also showed mechanical allodynia, increased alpha-2 macroglobulin levels and decreased thymus weight relative to the ”Healthy” cluster. These data provide preliminary support for a role of inflammation in decreasing psychological well-being after SCI. Overall, we demonstrate, post SCI, an interplay between decreased psychological well-being, increased peripheral inflammation and increased pain reactivity.

Are Serum Alpha-1-Antitrypsin and Alpha-2-Macroglobulin Levels Related with Tumor Histology or Stage of the Lung Cancer?.

In certain types of cancer, increased serum protease inhibitor levels were identified and clues showing that, serum levels of protease inhibitor could be used in the assessment of prognosis and dissemination of tumor were obtained. We aimed to reveal whether serum alpha-1-antitrypsin (AAT) and alpha-2-macroglobulin (AMG) levels of patients with lung cancer are related with histological type, clinical stage, and advance of cancer. The patients enrolled in the study were the ones with a histopathologic diagnosis of primary lung cancer and they were not administered any treatment before. Levels of serum AAT and AMG were analyzed at the moment of diagnosis. A total of 47 patients were enrolled in the study – 42 males and 5 females. The average age was 58.9. Tumor cell type was non-small cell lung carcinoma (NSCLC) in 36 and small cell lung carcinoma in 11 patients. There was a negative correlation between serum AAT level and age (p= 0.21 r=-0.339). No correlation was identified between serum AMG level and age (p= 0.138 r= 0.222). There was no significant relation between histological types of lung cancer and serums AAT and AMG (p= 0.807, p= 0.411 respectively). AAT and AMG levels were not different in limited and extensive small cell lung carcinoma (p= 0.201 and p= 0.465). AAT and AMG levels based on stages were not found to be different in NSCLC (p= 0.646 and p=0.650). AAT and AMG levels did not vary based on distant metastasis or local spread in NSCLC (p= 0.371 and p= 0.676). Serum alpha-1-antitrypsin and alpha-2-macroglobulin levels in patients with lung cancer do not vary based on histological types, stage of the disease, local, distant and lymphatic spread of the tumor.

Proenzyme therapy of sarcoma S-180 and melanoma B16-F10

The aim of this study was to evaluate the effectiveness of individual (inactive) proenzymes and mixtures thereof in cancer treatment and to compare this treatment with more frequently used therapy based on active proteases. Experiments focused on explanation of possible mechanisms of proenzyme action against tumours are included.Proenzyme therapy of sarcoma S-180 significantly reduced tumour growth and prolonged survival of mice. The effect of trypsinogen and chymotrypsinogen was synergistic. Proenzyme therapy of melanoma B16-F10 bearing mice reduced both tumour growth and prevalence of metastases. Active enzyme based therapy of melanoma B16-F10 was less effective. Severe combined immunodeficiency (SCID) mice bearing sarcoma S-180 did not respond to the proenzyme therapy, indicating that the effect of this therapy is dependent on fully developed acquired immunity. Measured decreased levels of TGF-β and an increased amount of alpha-2 macroglobulin in serum contributed to the elucidation of the cancer treatment mechanism.Proenzyme therapy based on administration of a mixture of trypsinogen and chymotrypsinogen is effective in cancer treatment.

Alpha2-macroglobulin as a promising biomarker for cerebral small vessel disease in acute ischemic stroke patients

Alpha2-macroglobulin is a protease inhibitor that enhances procoagulant properties via the neutralization of plasmin, plasminogen activators and metalloproteinases. Additionally, alpha2-macroglobulin is thought to be involved in inflammatory reactions as a carrier protein for interleukin-6 (IL-6). The objective of this study was to evaluate the usefulness of alpha2-macroglobulin as a biomarker for cerebrovascular diseases. Patients with acute ischemic stroke (n = 159; 93 male and 66 female, 71.6 ± 10.3 years) and patients with no previous history of stroke (n = 77; 38 male and 39 female, 70.7 ± 9.5 years) were consecutively enrolled in this study. White matter lesions were assessed via the fluid-attenuated inversion recovery image of magnetic resonance images using the Fazekas classification. The serum alpha2-macroglobulin levels were measured by nephelometry. The serum alpha2-macroglobulin levels at admission in patients with acute ischemic stroke were higher than those in the control patients (230.2 ± 73.7 vs. 205.0 ± 55.8 mg/dl, p = 0.009). The serum alpha2-macroglobulin levels were positively correlated with age and the severity of the white matter lesions (R (2) = 0.048, p < 0.001 and R (2) = 0.058, p < 0.001, respectively), although there was no significant association between serum alpha2-macroglobulin levels and IL-6 levels. In addition, multivariate analysis showed that increased serum alpha2-macroglobulin levels were independently associated with the severity of white matter lesions [standardized partial regression coefficient (β) 0.102, p = 0.026]. Increased serum alpha2-macroglobulin levels might be involved in the pathophysiology of acute ischemic stroke. Furthermore, serum alpha2-macroglobulin levels, which were associated with high-grade white matter lesions, may reflect the chronic pathophysiological condition of cerebral small vessel disease.

Complementary serum proteomic analysis of autoimmune hepatitis in mice and patients

BackgroundAutoimmune hepatitis (AIH) is a chronic liver disease caused by inflammation of the liver. The etiology of AIH remains elusive, and there are no reliable serum biomarkers.MethodsIn order to identify candidate biomarkers, 2-DE analysis of serum proteins was performed using a mouse model of AIH induced by treatment with concanavalin A (ConA). To enrich samples for low abundance molecules a commercial albumin removal reagent was used. In an independent analysis, candidate biomarkers were identified in AIH patient’s serum by a targeted iTRAQ (isobaric tags for relative and absolute quantification) identification. Candidates were validated in independent cohorts of ConA treated mice and AIH patients by ELISA (enzyme-linked immuno sorbent assay).ResultsNine proteins were differentially expressed in AIH mice treated with con-A. Two of these, the third component of complement (C3) and alpha-2-macroglobulin (A2M) were also up-regulated in AIH patient’s sera by a targeted iTRAQ identification. In separate validation studies, serum C3 and A2M levels were increased in mice with ConA treatment after 20-40 h and in 34 AIH patients in a subgroup analysis, females with AIH aged 20–50 years old displayed the largest increases in serum A2M level. Biological network analysis implements the complement cascade and protease inhibitors in the pathogenesis of AIH.ConclusionThe serum proteins C3 and A2M are increased both in a mouse model and in patients with AIH by both 2-DE and iTRAQ methods. This integrated serum proteomics investigation should be applicable for translational researchers to study other medical conditions.

Abstract TP226: Association Of Alpha2-macroglobulin As A Novel Biomarker With Acute Ischemic Stroke

Background: We have identified alpha2-macroglobulin as an acute stroke biomarker by using proteomic technologies with two-dimensional gel electrophoresis (2D-PAGE) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Alpha2-macroglobulin is known as a protease inhibitor, which enhance procoagulant properties by the neutralization of plasmin, plasminogen activators and metalloproteinases. The objective of this study was to evaluate the usefulness of serum alpha2-macroglobulin levels in acute ischemic stroke patients. Methods: Patients with acute ischemic stroke (n=159; 93 male and 66 female, 71.6±10.3 years) and patients with no previous stroke (n=77; 38 male and 39 female, 70.7±9.5 years) were consecutively enrolled in this study. Stroke subtype was evaluated based on the TOAST classification. Serum alpha2-macroglobulin levels were measured by nephelometry. Results: Serum alpha2-macroglobulin levels at admission in patients with acute ischemic stroke was higher than that in control patients (230.2±5.4 vs. 205.0±7.8 mg/dl, P=0.009). Higher age and white matter lesions by using Fazekas classification were significantly associated with high serum alpha2-macroglobulin levels (P=0.001 and P=0.005). After multivariate logistic regression analysis with sex, age, hypertension, diabetes mellitus, dyslipidemia, and atrial fibrillation, the highest quartile of serum alpha2-macroglobulin levels was independently associated with acute ischemic stroke (OR 1.52, 95% CI 1.13-2.08, P=0.005). Conclusion: Alpha2-macroglobulin, which was identified by using proteomics, was associated with higher age and white matter lesions. Additionally, serum alpha2-macroglobulin levels was associated with acute ischemic stroke independently from the other factors. Increased alpha2-macroglobulin may be involved in pathophysiology of acute ischemic stroke.

A preliminary study of inflammatory markers in non-alcoholic steatohepatitis patients

It was reported that C-reactive protein (CRP) levels increase in parallel with the progression of chronic liver diseases, such as chronic hepatitis and liver cirrhosis. Inflammatory markers, such as high sensitive C-reactive protein (hsCRP), ferritin, transferrin, albumin, alpha-1 acid glycoprotein (AAG), alpha-2 macroglobulin (AMG), alpha-1 anti-trypsin (AAT) and lipoprotein a [Lp(a)] were measured in coronary artery disease patients (CAD) and CAD patients with non-alcoholic steatohepatitis (NASH). In the present preliminary study an attempt was made to study whether there is an increase in the levels of CRP in CAD patients associated with NASH. CAD patients showed an increase in CRP and serum ferritin levels. In CAD patients with NASH along with an increase in the levels of serum ferittin (p<0.001), the levels of serum AMG and ceruloplasmin (CP) were also increased (p<0.01). The CAD patients with NASH had a higher proportion of diabetes, hypertension and dyslipidaemia compared to CAD patients. But how this difference contributes to the elevation in acute inflammatory markers particularly AMG and CP levels in CAD patients with NASH cannot be explained. This study shows that a substantial number of CAD patients may be associated with NASH. Non-invasive simple parameters that reflect the degree of inflammation and fibrosis of the liver in patients with NASH would facilitate improved understanding and treatment of the disease. Further studies may be necessary to evaluate the percentage of NASH patients progressing to CAD.

PAHA model: An alternative non‐invasive predictor of liver cirrhosis in patients with chronic hepatitis B infection

PAHA model: An alternative non‐invasive predictor of liver cirrhosis in patients with chronic hepatitis B infection

Lysosomal (leucocyte) proteinase and sulfatase levels in Dyggve-Melchior-Clausen (DMC) syndrome.

Patients with the DMC syndrome have been suggested to possess a specific sulfatase abnormality and/or to be deficient in a proteinase cleaving glycoprotein-acid mucopolysaccharide (AMP) linkage. We have previously found in DMC patients an abnormal excretion of urinary AMPs of which hyaluronic acid and chondroitin sulfate (A + C) were oversulfated and keratosulfate and heparan sulfate were undersulfated. Lysosomal acid proteinase, i.e. cathepsin D (EC 3.4.23.5) and neutral proteinase : elastase (EC 3.4.21.11) and cathepsin G were found to be normal in DMC patients. However, alpha 2-macroglobulin in serum was raised. This increase may be associated with a complex formation of alpha 2-macroglobulin with a neutral proteinase released from the cells. Increased levels of chondroitin sulfate N-acetylgalactosamine-6-sulfate sulfatase and sulfamidase and decreased enzymic levels of arylsulfatase A and B (EC 3.1.6.1) were found in leucocytes of DMC patients. The sulfatase activities assayed in the present study support our theory that a specific sulfatase abnormality may exist in the DMC syndrome.