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Simple and rapid detection of disease-related bio-markers are significant for early clinical diagnosis and can potentially improve the survival rate. However, establishing a high-specificity colorimetric detection method for bio-markers are still challenges due to their inevitable natural antibody used or enzymatic labeling. Herein, a cost-efficient and easy-to-use approach, which called dual molecular imprinting immunosandwich colorimetric strategy (DMI-ICS) was constructed for detection alpha-2-macroglobulin (α2MG) by janus imprinted nanoparticles. The unique detection principle was contained with two mimic antibody parts, the first part was α2MG glass slides molecularly imprinted material (GS-MIP) as a "Separation antibody", which can specifically rapid separate the protein in the complex sample; Another part was asymmetrically modified janus molecularly imprinted gold nanoparticles nanozyme (J-GNPs-MIP) as a "Detection antibody", which has the properties of specific recognition and catalytic substrate color performance at the same time. The concentration of α2MG can be determined by the substrate color changes and observed with naked eyes. Under the optimized conditions, the DMI-ICS had a great performance and offering lower relative standard deviation (RSD, 7.69%), good linear range (0.297-130 μg/mL, R 2  = 0.994), high imprinting factor (IF: 3.74) with lower detection limit (0.089 μg/mL). This strategy provides an easy operation and low cost signal readout method for direct detection and separation of α2MG in human serum samples, which is a versatile tool for point-of-care diagnosis, while also offering a new perspective on antibody simulation technology, multifunctional antibody preparation and contribute to detection of disease-related bio-marker in nonspecialized laboratory infrastructure., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

α-1-Acid glycoprotein (AGP) is an acute-phase protein that may suppress dry matter intake (DMI), potentially by acting on the leptin receptor in the hypothalamus. Our objectives were to characterize plasma AGP concentration and associations with DMI during the transition period, and to determine the utility of AGP to identify or predict cows with low DMI. Plasma samples (n = 2,086) from 434 Holstein cows in 6 studies were analyzed on d -21, -13 ± 2, -3, 1, 3, 7 ± 1, 14 ± 1, and 21 ± 1 relative to parturition. A commercially available ELISA kit specific for bovine AGP was validated, and 2 internal controls were analyzed on each plate with interplate variation of 15.0 and 17.3%, respectively. Bivariate analysis was used to assess the relationship between AGP and DMI. For significant associations, treatment(study) was added to the model, and quadratic associations were included in the model if significant. Plasma AGP concentration (±SEM) increased from 213 ± 37.3 μg/mL on d -3 to 445 ± 60.0 μg/mL on d 14. On d 3, AGP was associated negatively with DMI in a quadratic manner for wk 1 and wk 2 and linearly for wk 3. Day 7 AGP was associated negatively with DMI in a quadratic manner for wk 2 and linearly for wk 3. Similarly, d 14 AGP was negatively associated with DMI for wk 3 and wk 4. As d 3 AGP concentration increased over the interquartile range, a calculated 1.4 (8.5%), 0.5 (2.7%), and 0.4 (1.9%) kg/d reduction in predicted DMI was detected during wk 1, 2, and 3, respectively. Using bivariate analysis, d 3 AGP explained 10% of the variation in DMI during wk 1. We explored the clinical utility of d 3 AGP to diagnose low DMI, defined as wk 1 DMI >1 standard deviation below the mean. Receiver operating characteristic analysis identified a threshold of 480.9 μg/mL, providing 76% specificity and 48% sensitivity (area under the curve = 0.60). Limited associations occurred between AGP and blood biomarkers; however, AGP was associated with plasma haptoglobin concentration postpartum and incidence of displaced abomasum, retained placenta, and metritis. These results demonstrate a negative association between plasma AGP concentration and DMI in early-postpartum dairy cows, although its diagnostic performance was marginal. Further investigation into whether AGP directly suppresses DMI in dairy cattle is warranted., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Background: Non-invasive tests that can identify patients with non-alcoholic steatohepatitis (NASH) at higher risk of disease progression are lacking. We report the development and validation of a blood-based diagnostic test to non-invasively rule in and rule out at-risk NASH (defined as non-alcoholic fatty liver disease [NAFLD] activity score [NAS] ≥4 and fibrosis stage ≥2)., Methods: In this prospective derivation and global validation study, blood samples, clinical data, and liver biopsy results from three independent cohorts with suspected NAFLD were used to develop and validate a non-invasive blood-based diagnostic test, called NIS4. Derivation was done in the discovery cohort, which comprised 239 prospectively recruited patients with biopsy-confirmed NASH (NAFLD NAS ≥3; fibrosis stage 0-3) from the international GOLDEN-505 phase 2b clinical trial. A complete matrix based on 23 variables selected for univariate association with the presence of at-risk NASH and avoiding high multi-collinearity was used to derive the model in a bootstrap-based process that minimised the Akaike information criterion. The overall diagnostic performance of NIS4 was externally validated in two independent cohorts: RESOLVE-IT diag and Angers. The RESOLVE-IT diag cohort comprised the first 475 patients screened for potential inclusion into the RESOLVE-IT phase 3 clinical trial. Angers was a retrospective cohort of 227 prospectively recruited patients with suspected NAFLD and clinical risk factors for NASH or fibrosis stage 2 or more according to abnormal elastography results or abnormal liver biochemistry. Both external validation cohorts were independently analysed and were combined into a pooled validation cohort (n=702) to assess clinical performance of NIS4 and other non-invasive tests., Findings: The derived NIS4 algorithm comprised four independent NASH-associated biomarkers (miR-34a-5p, alpha-2 macroglobulin, YKL-40, and glycated haemoglobin; area under the receiver operating characteristics curve [AUROC] 0·80, 95% CI 0·73-0·85), and did not require adjustment for age, sex, body-mass index (BMI), or aminotransferase concentrations. Clinical cutoffs were established within the discovery cohort to optimise both rule out and rule in clinical performance while minimising indeterminate results. NIS4 was validated in the RESOLVE-IT diag cohort (AUROC 0·83, 95% CI 0·79-0·86) and the Angers cohort (0·76, 0·69-0·82). In the pooled validation cohort, patients with a NIS4 value less than 0·36 were classified as not having at-risk NASH (ruled out) with 81·5% (95% CI 76·9-85·3) sensitivity, 63·0% (57·8-68·0) specificity, and a negative predictive value of 77·9% (72·5-82·4), whereas those with a NIS4 value of more than 0·63 were classified as having at-risk NASH (ruled in) with 87·1% (83·1-90·3) specificity, 50·7% (45·3-56·1) sensitivity, and a positive predictive value of 79·2% (73·1-84·2). The diagnostic performance of NIS4 within the external validation cohorts was not influenced by age, sex, BMI, or aminotransferase concentrations., Interpretation: NIS4 is a novel blood-based diagnostic that provides an effective way to non-invasively rule in or rule out at-risk NASH in patients with metabolic risk factors and suspected disease. Use of NIS4 in clinical trials or in the clinic has the potential to greatly reduce unnecessary liver biopsies in patients with lower risk of disease progression., Funding: Genfit., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Benralizumab and mepolizumab are new therapies for severe eosinophilic asthma. They are both humanized IgG antibodies, targeting the IL-5 receptor and IL-5, respectively, suppressing the corresponding pathways. No specific biomarkers have been proposed to evaluate treatment response to benralizumab or mepolizumab. The aim of this proteomic study was to compare serum protein profiles of patients with severe eosinophilic asthma before and after anti-IL5 or anti-IL5R therapies. Proteomic analysis highlighted 22 differently abundant spots. Among the proteins identified, CAYP1, A1AT and A2M expression was significantly modified in both groups of patients after therapies while ceruloplasmin showed a significant modification in the group of benralizumab treatment. These differentially expressed proteins could be potential biomarkers of response to mepolizumab and benralizumab treatments and need further evaluation.

While pregnancy-related proteins (PRP) are known to contribute to immunotolerance during pregnancy, their significance to development of invasive placenta is unclear. We compared PRP expression in humans and the common marmoset (Callithrix jacchus), a new-world monkey. Invasive placenta was observed at the maternal-foetal interface of marmoset placenta from green fluorescent protein (GFP)-expressing foetus and wild type mother. The pregnancy zone protein (PZP) and alpha-2 macroglobulin-like 1 (A2ML1) proteins exhibited the most prominent increase in expression during the second trimester in humans and marmoset, respectively. In humans, PZP accumulated at the maternal-foetal interface and A2ML1 accumulated in the amnion. Similarly, A2ML1 mRNA was detected in marmoset placenta. These proteins belong to the A2M family of protease inhibitors, and both PZP and A2ML1 share around 90% homology between human and marmoset and have highly conserved structures. However, the protease-reacting bait regions of the proteins had lower homology (56.8-60.7% in proteins) relative to the rest of the sequence. Notably, the cleavage site of a proinflammatory proline-endopeptidase was preserved in human PZP and marmoset A2ML1. These proteins contain multiple sites that are cleaved by proteases involving proline-endopeptidase. Systemic regulation of these A2M family proteins may be important in animals with invasive placenta.

Noninvasive biomarkers are clinically useful for evaluating liver fibrosis stage in patients with nonalcoholic fatty liver disease (NAFLD). The aim of the present study was to compare plasma proteins in patients with early nonalcoholic steatohepatitis (NASH) (F0-F1) versus NASH with significant/advanced fibrosis (F2-F4) to determine whether candidate proteins could be used as potential noninvasive biomarkers. Nineteen biopsy-proven NAFLD patients including ten early NASH patients and nine NASH patients with significant/advanced fibrosis were enrolled in the present study. High-resolution proteomics screening of plasma was performed with the SCIEX TripleTOF 5600 System. Proteins were quantified using two different software platforms, Progenesis Qi and Scaffold Q+, respectively. Progenesis Qi analysis resulted in the discovery of 277 proteins compared with 235 proteins in Scaffold Q+. Five consensus proteins (i.e. Complement component C7; α-2-macroglobulin; Complement component C8 γ chain; Fibulin-1; α-1-antichymotrypsin) were identified. Complement component C7 was three-fold higher in the NASH group with significant/advanced fibrosis (F2-F4) compared with the early NASH (F0-F1) group (q-value = 3.6E-6). Complement component C7 and Fibulin-1 are positively correlated with liver stiffness (P=0.000, P=0.002, respectively); whereas, Complement component C8 γ chain is negatively correlated (P=0.009). High levels of Complement C7 are associated with NASH with significant/advanced fibrosis and Complement C7 is a perfect classifier of patients included in this pilot study. Further studies will be needed in a larger validation cohort to confirm the utility of complement proteins as biomarkers or mechanistic determinants of NASH with significant/advanced fibrosis., (© 2020 The Author(s).)

Objectives: Sepsis is associated with high early and total in-hospital mortality. Despite recent revisions in the diagnostic criteria for sepsis that sought to improve predictive validity for mortality, it remains difficult to identify patients at greatest risk of death. We compared the utility of nine biomarkers to predict mortality in subjects with clinically suspected bacterial sepsis., Design: Cohort study., Setting: The medical and surgical ICUs at an academic medical center., Subjects: We enrolled 139 subjects who met two or more systemic inflammatory response syndrome (systemic inflammatory response syndrome) criteria and received new broad-spectrum antibacterial therapy., Interventions: We assayed nine biomarkers (α-2 macroglobulin, C-reactive protein, ferritin, fibrinogen, haptoglobin, procalcitonin, serum amyloid A, serum amyloid P, and tissue plasminogen activator) at onset of suspected sepsis and 24, 48, and 72 hours thereafter. We compared biomarkers between groups based on both 14-day and total in-hospital mortality and evaluated the predictive validity of single and paired biomarkers via area under the receiver operating characteristic curve., Measurements and Main Results: Fourteen-day mortality was 12.9%, and total in-hospital mortality was 29.5%. Serum amyloid P was significantly lower (4/4 timepoints) and tissue plasminogen activator significantly higher (3/4 timepoints) in the 14-day mortality group, and the same pattern held for total in-hospital mortality (Wilcoxon p ≤ 0.046 for all timepoints). Serum amyloid P and tissue plasminogen activator demonstrated the best individual predictive performance for mortality, and combinations of biomarkers including serum amyloid P and tissue plasminogen activator achieved greater predictive performance (area under the receiver operating characteristic curve > 0.76 for 14-d and 0.74 for total mortality)., Conclusions: Combined biomarkers predict risk for 14-day and total mortality among subjects with suspected sepsis. Serum amyloid P and tissue plasminogen activator demonstrated the best discriminatory ability in this cohort.

To develop a mathematical model and evaluate its prognostic significance for determining the severity of attack (SA) in patients with ulcerative colitis (UC) on the basis of minimally invasive laboratory inflammatory tests and albumin. The study involved 64 patients (33 men and 31 women) with UC in the stage of active inflammation. The method for diagnosing SA was performed by determining the concentration in the blood of tumor necrosis factor alpha (TNF-α), C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), α2-globulin and albumin. Truelove and Witts criteria (1955) were used as a reference method for the evaluation of SA in patients with UC. In constructing a mathematical model, the severity of the attack of the UC emerged as a dependent variable. As independent variables or predictors - five laboratory parameters: TNF-α, CRP, ESR, α2-globulins and albumin, which had a high relationship with SA. At SA value in the range from 0 to 0.5, absence of attack or remission of the disease is determined, SA value in the range from 0.5 to 1.2 corresponds to mild severity of UC attack, SA value in the range from 1.2 to 2.2 corresponds to the moderate severity of the attack of the UC, at SA more than 2.2 one should speak of a severe attack of the UC. The model is statistically significant: the Fisher test F = 439.9; p < 0.0001, multiple R = 0.981; R2 = 0.961. The diagnostic sensitivity of this mathematical model was 98.4%, specificity - 96.7%. The index of the severity of the attack, calculated by the formula proposed by us, is able to differentiate the mild, moderate and severe SA, including the isolation of patients with a stage of remission. In this case, the method is non-traumatic, has a low cost and high sensitivity and specificity., Competing Interests: The authors declare no conflict of interest.

Osteonecrosis of the femoral head (ONFH) is a potentially devastating complication that occurs in up to 40% of young adults receiving chronic glucocorticoid (GC) therapy. Through a validated GC therapy rat model, we have previously shown that Wistar Kyoto (WK) rats exhibit a genetic susceptibility to GC-induced ONFH compared to Sasco Fischer (F344) rats. We have undertaken this study in order to investigate differences between these two strains for their bone parameters, alpha-2-macroglobulin (A2M) circulating levels and incidence of GC-induced osteonecrosis of the femoral head. WK and F344 rats were treated either with 1.5 mg/kg/day of prednisone or placebo for 6 months. Blood was taken every month. The femoral heads were harvested for histological examination to detect ONFH and analyzed with micro-computed tomography. After 3 months of GC-therapy, plasma A2M was elevated in treated rats only. GC-treated WK rats exhibited histological evidence of early ONFH through higher rates of cellular apoptosis and empty osteocyte lacunae in the subchondral bone compared to placebos and to F344 rats. Furthermore, micro-CT analysis exhibited femoral head collapse only in GC-treated WK rats. Interestingly, GC-treated F344 rats exhibited significant micro-CT changes, but such changes were less concentrated in the articular region and were accompanied histologically with increased marrow fat. These µCT and histological findings suggest that elevated A2M serum level is not predictive and suitable as an indicative biomarker for early GC-induced ONFH in rodents. Elevated A2M levels observed during GC treatment suggests that it plays role in the host reparative response to GC-associated effects. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1183-1194, 2017., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Aims: The aims of this study were to increase the diagnostic accuracy of the analysis of synovial fluid in the differentiation of prosthetic joint infection (PJI) by the addition of inexpensive biomarkers such as the levels of C-reactive protein (CRP), adenosine deaminase (ADA), alpha-2-macrogloblulin (α2M) and procalcitonin., Patients and Methods: Between January 2013 and December 2015, synovial fluid and removed implants were requested from 143 revision total joint arthroplasties. A total of 55 patients met inclusion criteria of the receipt of sufficient synovial fluid, tissue samples and removed implants for analysis. The diagnosis of PJI followed the definition from a recent International Consensus Meeting to create two groups of patients; septic and aseptic. Using receiver operating characteristic curves we determined the cutoff values and diagnostic accuracy for each marker., Results: There were 23 PJIs and 32 patients with aseptic loosening. The levels of total leucocyte count, proportion of polymorphonuclear leucocytes (PMNs), CRP, ADA and α2M in the synovial fluid were all significantly higher in those with a PJI than in those with aseptic loosening. The levels of procalcitonin were comparable in the two groups. Cutoff values for the optimal performance in the diagnosis of infection were: total leucocyte count > 1463 cells/μL (sensitivity (Sens) 100%, specificity (Spec) 71.9%, positive predictive value (PPV) 71.9%, negative predictive value (NPV) 100%); proportion of PMNs > 81% (Sens 78.3%, Spec 75.0%, PPV 69.2%, NPV 82.8%); CRP > 6.7mg/L (Sens 78.3%, Spec 93.8%, PPV 90.0%, NPV 85.7%); ADA > 61U/L (Sens 78.3%, Spec 96.9%, PPV 94.7%, NPV 86.1%) and α2M > 958 mg/L (Sens 47.8%, Spec 96.9%, PPV 91.7%, NPV 72.1%). The addition of a raised level of CRP or ADA to the total leukocyte count increased the specificity: total leukocyte count > 1463 cells/μL and CRP > 6.7mg/L (Sens 78.3%, Spec 100%, PPV 100%, NPV 86.5%) or with ADA > 61U/L (Sens 78.3%, Spec 96.9%, PPV 94.7%, NPV 86.1%)., Conclusion: The total leucocyte count in the synovial fluid offers great negative predictive value in the diagnosis of PJI and the addition of more specific markers such as CRP and ADA improves the positive predictive value. Thus the addition of simple and inexpensive markers to the measurement of the leucocyte count in the synovial fluid may reduce the number of equivocal results which demand more expensive investigation. Cite this article: Bone Joint J 2017;99-B:351-7., (©2017 The British Editorial Society of Bone & Joint Surgery.)

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.

Unlabelled: The early diagnosis and successful treatment of breast cancer (BC) is still a challenging task due to the diverse origin and functional heterogeneity of cancer cells. The heterogeneity of BC may likely to explained by molecular BC subtypes, comprises Luminal-A (LA), Luminal-B (LB), Triple-negative (TN) and HER2-positive (HP), which are governed by a variety of cancer associated pathways. To identify protein signatures in different BC subtypes, we performed isobaric tag for absolute and relative quantitation (iTRAQ) of enriched blood plasma samples of BC subtypes (N=32) and healthy subjects (N=8). After analyses of data, 58 proteins were found to be modulated in BC subtypes from healthy subjects (p<0.05) and among these; Fibronectin (FN1), Alpha-2-macroglobulin (A2M), and Complement component-4-binding protein-alpha (C4BPA) and Complement factor-B (CFB) were selected for validation in BC subtypes and healthy subjects in the independent set of blood plasma (N=100) and tissue samples (N=25). Statistical analysis showed the significant modulation of FN1 and C4BPA in LB, and A2M in TN patients in both plasma as well as tissues comparatively control (p<0.05). Further, FN1 and C4BPA in LB subtype revealed a good diagnostic accuracy in plasma level validation. The receiver operating characteristic (ROC) curve and regression analysis demonstrated that these proteins with associated criterion of expression could act as discriminating signatures among BC subtypes with diagnostic and prognostic relevance., Significance: The heterogeneity of breast cancer (BC) has gained many challenges for successful management of BC, thus, the delineating proteomic alterations BC subtypes may provide great clinical values in diagnostic, prognostic and therapeutics of BC. The findings from the present quantitative proteomic study have deciphered the altered proteomic patterns and their possible molecular interactions in each BC subtype. The study showed a strong association of FN1, A2M, C4BPA and CFB in molecular subtypes of BC, in which, C4BPA and A2M demonstrated a potent signature in blood plasma and tissue samples of LB and TN subtypes in BC patients, respectively. The findings also revealed the altered level expressions of these selected proteins could classify BC subtypes through plasma and tissue based expression analysis in patients and control samples. Hence, these proteins could have clinical importance for the diagnosis and prognosis purposes among molecular BC subtypes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

As an abundant plasma protein, α2-macroglobulin (α2-M) participates widely in physiological and pathological activities including coagulation regulation, antitumor activities, and regulation of cytokines. It also presents a therapeutic potential for radiation injury. A two-step isolation method for the purification of α2-M from Cohn Fraction IV is described. This process includes a salting-out method and immobilized metal affinity chromatography. The LC-ESI-MS/MS analysis and a comparison of the amino acid composition demonstrated that the final product was α2-M. The final protein, with a purity of approximately 95% and a yield of nearly 45%, was obtained from Cohn Fraction IV regardless of plasma haptoglobin type, although all but type 1-1 have previously been considered unfavorable for α2-M preparation. The effects of temperature, pH, and methylamine on α2-M activity were evaluated to avoid activity loss during preparation and preservation. The results suggested that α2-M activity could be readily inactivated at temperatures above 50°C, at pH levels above 9.0 or below 4.0, or in the presence of methylamine. Cohn Fraction IV is usually discarded as a biological waste product in the human serum albumin production process; because the simple process developed in this study is relatively inexpensive, the preparation of α2-M from Cohn Fraction IV may better utilize human plasma, a valuable resource., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: There is an urgent need for new tools for the rapid diagnosis of tuberculosis (TB) disease in resource-constrained settings. Tests based on host immunological biomarkers maybe useful, especially if based on easily available samples. We investigated host biomarkers detected in saliva samples from individuals with suspected pulmonary TB disease, as tools for the diagnosis of TB disease and monitoring of the response to treatment., Methods: We collected saliva samples from 104 individuals that presented with symptoms requiring investigation for TB disease at a primary health care clinic in the outskirts of Cape Town, South Africa, prior to assessment for TB disease. We evaluated the concentrations of 33 host markers in stored saliva samples using a multiplex cytokine platform. Using a combination of clinical, radiological and laboratory results and a pre-established diagnostic algorithm, participants were later classified as having TB disease or other respiratory diseases (ORD). The diagnostic potentials of individual analytes were analysed by the receiver operator characteristics curve approach while the predictive abilities of combinations of analytes for TB disease were analysed by general discriminant analysis, with leave-one-out cross validation., Results: Of the 104 individuals enrolled, 32 were pulmonary TB cases. There were significant differences in the levels of 10 of the markers investigated between the patients with TB disease and those with ORDs. However, the optimal diagnostic biosignature was a seven-marker combination of salivary CRP, ferritin, serum amyloid P, MCP-1, alpha-2-macroglobulin, fibrinogen and tissue plasminogen activator. This biosignature diagnosed TB disease with a sensitivity of 78.1% (95% CI, 59.6-90.1%) and specificity of 83.3% (95% CI, 72.3-90.7%) after leave-one-out cross validation. When compared to baseline levels, the concentrations of 9 markers including granzyme A, MCP-1, IL-1β, IL-9, IL-10, IL-15, MIP-1β, ferritin and serum amyloid A changed significantly by months 2 or 6 after initiation of TB treatment, thereby indicating that they might be useful in monitoring the response to TB treatment., Conclusion: We have identified candidate biomarkers in saliva, which may be useful in the diagnosis of TB disease and monitoring of the response to TB treatment. These results require further validation in larger studies., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Increasingly, clinical trials for Alzheimer's disease (AD) are being conducted earlier in the disease phase and with biomarker confirmation using in vivo amyloid PET imaging or CSF tau and Aβ measures to quantify pathology. However, making such a pre-clinical AD diagnosis is relatively costly and the screening failure rate is likely to be high. Having a blood-based marker that would reduce such costs and accelerate clinical trials through identifying potential participants with likely pre-clinical AD would be a substantial advance. In order to seek such a candidate biomarker, discovery phase proteomic analyses using 2DGE and gel-free LC-MS/MS for high and low molecular weight analytes were conducted on longitudinal plasma samples collected over a 12-year period from non-demented older individuals who exhibited a range of 11C-PiB PET measures of amyloid load. We then sought to extend our discovery findings by investigating whether our candidate biomarkers were also associated with brain amyloid burden in disease, in an independent cohort. Seven plasma proteins, including A2M, Apo-A1, and multiple complement proteins, were identified as pre-clinical biomarkers of amyloid burden and were consistent across three time points (p <  0.05). Five of these proteins also correlated with brain amyloid measures at different stages of the disease (q <  0.1). Here we show that it is possible to detect a plasma based biomarker signature indicative of AD pathology at a stage long before the onset of clinical disease manifestation. As in previous studies, acute phase reactants and inflammatory markers dominate this signature.

The integrity of the fetal amnion-chorion is an imperative for the preservation of a normal pregnancy in the human. The diagnosis of the status of the fetal membranes has traditionally been reduced to either intact or ruptured. In the last decades, evidence has accumulated demonstrating that this clinical approach may well be an over simplification. Practically, all maternal organs experienced physiologic or eventually pathologic changes during the length of the gestational period. We propose that the fetal membranes are also significantly impacted by those changes. The accurate, specific, simplified and low-cost diagnosis of the status of the fetal membranes is of critical importance for the assessment of risk to the pregnancy followed by efficient and prompt treatment. The presence of placental alpha macroglobulin-1 in the vagina specifically indicates a disruption in the integrity of the fetal membranes and may indirectly mean increased risk for preterm birth. Further research to properly characterize this marker and its importance in the care of pregnant woman at risk for preterm birth is strongly recommended.

An elevated serum albumin (ALB) and albumin/globulin ratio (AGR) have been reported to be associated with a favorable prognosis for several types of cancer. However, little is known about prognostic significance of globulin (GLB) in gastric cancer patients. The purpose of this study was to evaluate whether GLB, ALB, and AGR analysis could predict the prognosis of patients with gastric cancer. A retrospective cohort of 186 patients with gastric cancer followed by radical surgery was recruited between January 2007 and December 2010. Levels for preoperative GLB and ALB were obtained and used to calculate the AGR. Survival analysis was used to evaluate the predictive value of GLB, ALB, and AGR. X-tile program determined 37.6, 33.4, and 1.33 as the optimal cutoff value for ALB, GLB, and AGR in terms of survival. Univariate analysis revealed that low GLB levels were significantly associated with favorable survival (P = 0.045). Conversely, low ALB levels were associated with a significantly worse survival (P = 0.000). In conclusion, low preoperative GLB level may serve as a valuable marker to predict the prognosis of gastric cancer patients.

Aim: To analyze whether comprehensive assessment of haemostatic system components, in particular, indices of coagulation and fibrinolytic systems along with functionally related proteins, could be indicative of upper respiratory tract (URT) cancer progression., Materials and Methods: Indices of coagulation and fibrinolytic systems along with functionally related proteins, in particular, trypsin-like amidolytic activity, trypsin-like proteolytic activity, thrombin-like amidolytic activity, elastase-like amidolytic activity, fibrinolytic activity, potential amidolytic plasmin activity, content of fibrinogen, antithrombin III, α1-proteinase inhibitor, and α2-macroglobulin, and prothrombin time were evaluated in blood plasma of patients with URT cancer of II (n = 10) and III (n = 25) stages with the use of routine biochemical methods., Results: For both groups of patients with URT cancer there have been shown notable differences for the majority of the studied indices, especially the indexes of proteolytic activities, from these of healthy donors, and in the case of URT cancer of III stage they reached statistical significance. In contrary, the changes in the content of antithrombin III, α1-proteinase inhibitor, and α2-macroglobulin were insignificant. In both groups of patients significant increase of fibrinogen content has been registered, while the content of soluble fibrinogen didn't change. Also, in both groups of patients there a significant increase of potential activity of plasminogen was documented, while clot lysis time was significantly increased only in patients with III stage URT cancer. Multifactorial analysis of haemostatic system indices evidenced for efficacy of their combined use for evaluation of URT cancer progression risk., Conclusion: Combined use of fibrinogen and α2-macroglobulin content and the level of amidolytic thrombin-like activity could serve as an indicator of URT cancer progression.

Background: The bromocresol green (BCG) assay is commonly used for measuring albumin (ALB), but is affected by α1- and α2-globulins, which are elevated in systemic inflammation. The modified bromocresol purple (mBCP) assay is another dye-binding method developed to overcome non-specific reactions. Concentrations of α2-macroglobulin, a major α2-globulin component, are increased in nephrotic syndrome (NS), but not in inflammation. There is little direct evidence that α2-macroglobulin affects BCG or mBCP assays., Methods: We measured serum albumin concentrations in 33 patients with NS and 13 reference healthy controls using BCG (ALBBCG) and mBCP (ALBmBCP) assays, and nephelometry (nALB) as a reference method. We also determined five specific proteins belonging to the α1- and α2-globulins by nephelometry. After adding purified α2-macroglobulin to albumin solutions, protein reactivity in these three assays was compared., Results: Nephrotic syndrome patients were categorized to tertiles according to nALB concentration. In all tertiles, ALBBCG was significantly higher than nALB, especially in the severe hypoalbuminemia group, in which α2-macroglobulin was 43-49% higher. By contrast, ALBmBCP and nALB were almost identical in all three groups. The difference between ALBBCG and nALB was positively correlated with the α2-macroglobulin concentration. In vitro, when α2-macroglobulin was added to solutions containing identical albumin concentrations, α2-macroglobulin dose-dependently increased ALBBCG, but not ALBmBCP., Conclusions: In NS, α2-macroglobulin is a major factor for positive bias of ALBBCG, especially in patients with severe hypoalbuminemia. The mBCP assay is useful for measuring albumin concentrations in NS., (© The Author(s) 2015.)

Lack of diagnostic capacity has been a crucial barrier preventing an effective response to the challenges of malnutrition and tuberculosis (TB). Point-of-care diagnostic tests for TB in immuno-incompetent, malnourished population are thus needed to ensure rapid and accurate detection. The aim of the study was to identify potential biomarkers specific for TB infection and progression to overt disease in the malnourished population of Melghat. A prospective cohort study was conducted in the year 2009 through 2011 in six villages of the Melghat region. 275 participants consisting of malnourished cases with a) active TB (n = 32), b) latent TB infection (n = 90), c) with no clinical or bacteriological signs of active or latent TB (n = 130) and healthy control subjects (n = 23) were recruited for the study. The proteome changes of the host serum in response to Mycobacterium tuberculosis (M.tb) infection were investigated using one dimensional electrophoresis in combination with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Three most differentially expressed proteins; alpha-2-macroglobulin (A-2-M), sero-transferrin and haptoglobin were identified by MALDI-TOF MS analysis, which were up-regulated in the malnourished patients with active TB and down-regulated in the malnourished patients compared with the healthy controls. Additionally, follow-up studies indicated that the expression of these proteins increased to nearly two folds in patients who developed active disease from latent state. Our preliminary results suggest that A-2-M, sero-transferrin and haptoglobin may be clinically relevant host biomarkers for TB diagnosis and disease progression in the malnourished population. This study provides preliminary framework for an in-depth analysis of the biomarkers in larger well-characterized cohorts. Evaluation of these biomarkers in follow-up cases may further aid in improving TB diagnosis.

Alzheimer's disease (EOAD, LOAD), mild cognitive impairment (MCI), Parkinson's disease (PD) and healthy controls were included to determine the serum interleukin-1s (IL-1α, IL-1β), IL-6 and alpha-2-macroglobulin (α2M) levels using ELISA. IL-6 might be a significant contributor to the inflammatory response in LOAD. The MCI data indicate that IL-1s, α2M and BDNF are somehow related, and this relationship might allow MCI patients to be more similar to the healthy controls. A correlation analysis of multiple biomarkers in different neurodegenerative disorders might be more useful than determining the levels of a single cytokine in a single disorder., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Objectives: Dengue virus (DENV) infection is a significant risk to over a third of the human population that causes a wide spectrum of illness, ranging from sub-clinical disease to intermediate syndrome of vascular complications called dengue fever complicated (DFC) and severe, dengue hemorrhagic fever (DHF). Methods for discriminating outcomes will impact clinical trials and understanding disease pathophysiology., Study Design: We integrated a proteomics discovery pipeline with a heuristics approach to develop a molecular classifier to identify an intermediate phenotype of DENV-3 infectious outcome., Results: 121 differentially expressed proteins were identified in plasma from DHF vs dengue fever (DF), and informative candidates were selected using nonparametric statistics. These were combined with markers that measure complement activation, acute phase response, cellular leak, granulocyte differentiation and viral load. From this, we applied quantitative proteomics to select a 15 member panel of proteins that accurately predicted DF, DHF, and DFC using a random forest classifier. The classifier primarily relied on acute phase (A2M), complement (CFD), platelet counts and cellular leak (TPM4) to produce an 86% accuracy of prediction with an area under the receiver operating curve of >0.9 for DHF and DFC vs DF., Conclusions: Integrating discovery and heuristic approaches to sample distinct pathophysiological processes is a powerful approach in infectious disease. Early detection of intermediate outcomes of DENV-3 will speed clinical trials evaluating vaccines or drug interventions., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Apolipoprotein A-I (Apo A-I) is a major protein in lipid/lipoprotein metabolism and decreased serum levels have been observed in many species in response to inflammatory and infectious challenges. Little is known about the porcine homologue, therefore in this work we have characterized it through biochemical and proteomic techniques. In 2DE, porcine serum Apo A-I is found as three spots, the two more acidic ones corresponding to the mature protein, the more basic spot to the protein precursor. Despite high sequence coverage in LC-MS/MS, we did not find a sequence or PTM difference between the two mature protein species. Besides this biochemical characterization, we measured overall levels and relative species abundance of serum Apo A-I in four different viral and bacterial porcine infectious diseases. Lower overall amounts of Apo A-I were observed in Salmonella typhimurium and Escherichia coli infections. In the 2DE protein pattern, an increase of the protein precursor together with a lower level of mature protein species were detected in the porcine circovirus type 2-systemic disease and S. typhimurium infection. These results reveal that both the porcine serum Apo A-I concentration and the species pattern are influenced by the nature of the infectious disease., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Increased serum concentrations of homocysteine (HCY) and methylmalonic acid (MMA), the 2 main cobalamin-dependent metabolites, as well as decreased serum albumin and canine alpha1 -proteinase inhibitor (cα1 -PI) concentrations have previously been described in hypocobalaminemic dogs with gastrointestinal disease. However, no studies have been conducted to evaluate potential relationships between these serum biomarkers., Objective: The aim of this study was to evaluate the relationship between HCY and MMA, 2 cobalamin-dependent metabolites, and both serum albumin and cα1 -PI concentrations in hypocobalaminemic dogs., Methods: Serum samples from 285 dogs including 7 different breeds (Beagle, Boxer, Cocker Spaniel, German Shepherd, Labrador Retriever, Chinese Shar-Pei, and Yorkshire Terrier) with hypocobalaminemia were used. Serum HCY, MMA, albumin, and cα1 -PI concentrations were determined., Results: There was a significant correlation between serum HCY and albumin concentrations, as well as serum HCY and cα1 -PI concentrations (ρ = 0.62 and ρ = 0.37, respectively; P < .0001). No correlations were observed between serum MMA and albumin concentrations, or cα1 -PI concentrations (ρ = 0.01 and ρ = 0.08, respectively; P > .05). In addition, significant breed-specific correlations were observed between serum MMA and albumin concentrations in German Shepherds, and serum HCY and MMA concentrations in Chinese Shar-Peis with hypocobalaminemia., Conclusions: This study shows a correlation between serum albumin and cα1 -PI and HCY concentrations, but not with serum MMA concentration in dogs with hypocobalaminemia. In addition, significant breed-specific correlations were observed between serum MMA and albumin concentrations in German Shepherds, as well as serum HCY and MMA concentrations in Chinese Shar-Peis, emphasizing the unique metabolic interactions in those dog breeds affected by hypocobalaminemia., (© 2014 American Society for Veterinary Clinical Pathology.)

Aging is considered to be an adaptive mechanism to altered needs of an organism and/or to altered stimuli. Plasma concentrations of insulin-like growth factor binding protein-2 (IGFBP-2) increase with age and it is generally assumed that IGFBP-2 is a negative predictor of healthy aging. The aim of this study was to examine the distribution of IGFBP-2 molecular forms in different age groups and, specifically, the relationship between IGFBP-2 and alpha-2-macroglobulin (α2M). The relative amount of monomer IGFBP-2 was the highest in young persons, making up approximately 2/3 of the total circulating IGFBP-2. This gradually decreased with age down to 1/3 of total IGFBP-2 in elderly individuals. Fragmented IGFBP-2 increased with age and contributed almost 60% to the total immunoreactive IGFBP-2 in the age group 61-80 years. IGFBP-2/α2M complexes represented 10-12% of the total IGFBP-2 in the two younger groups but half this level in the oldest group. The significance of these changes and whether they affect more IGF-dependent or independent interactions are unknown. Due to drastic proteolysis of IGFBP-2, it may be postulated that either over-release of IGFBP-2-bound IGFs causes unwanted events or IGFBP-2 fragments are able to over-stimulate cellular processes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: A revised classification of chronic kidney disease (CKD) was proposed by the Kidney Disease: Improving Global Outcomes (KDIGO) in 2012. Neutrophil gelatinase-associated lipocalin (NGAL) was considered as one of the most promising biomarkers in clinical nephrology. The aim of this study was to examine the level of NGAL in patients with different impairment of GFR based on the new classification, and to evaluate whether NGAL in serum or urine was associated with different risk categories in CKD patients., Methods: A cross-sectional study was performed in 240 patients with CKD. NGAL, serum cystatin C, β₂-macroglobulin (β₂-MG), urine α₁-macroglobulin (α₁-MG) and albuminuria were tested in patients with various degrees of renal impairment., Results: Good correlation was found between the NGAL and the cystatin C, β₂-MG and the α₁-MG (r > 0.7). The level of sNGAL in CKD stage 3b was more than that in CKD stage 3a (P = 0.025). The concentration of the NGAL increased progressively with the increasing of risk categories (proposed by the revised CKD classification). The cutoff value of NGAL was calculated from stage 2 to stage 5. ROC analysis showed good AUC (sNGAL > 0.8, uNGAL > 0.7) and high specificity (sNGAL > 87%, uNGAL > 90%) on the cutoff value of NGAL., Conclusion: The results confirm NGAL as a useful biomarker in clinical nephrology which is helpful to diagnosis and evaluate the categories for CKD proposed by the KDIGO.

We describe a label-free relative quantification LC-MS/MS method for core-fucosylation in alpha-2-macroglobulin (A2MG) immunoprecipitated from human sera. The method utilizes endoglycosidase F partial deglycosylation to reduce glycosylation microheterogeneity, while retaining the innermost N-acetylglucosamine (GlcNAc) and core fucose. Precursor ion peak areas of partially deglycosylated peptides were obtained and site-specific core-fucosylation ratios based on the peak areas of core-fucosylated and nonfucosylated counterparts were calculated and evaluated for assay development. This assay was applied in a preliminary study of sera samples from normal controls and patients with pancreatic diseases, including pancreatic cancer and chronic pancreatitis. A2MG fucosylation levels at sites N396 and N1424 were found to decrease in both chronic pancreatitis and pancreatic cancer compared to normal controls. The two sites were identified by two peptides and their core-fucosylation ratios were found to be internally consistent. This method provides a platform to quantify fucosylation levels and can be used to study site-specific core-fucosylation aberrations in other glycoproteins for other diseases., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background and Aims: The PGAA index was one of the first composite liver fibrosis markers. This study aims, prospectively, to confirm the diagnostic value of PGAA and Fibrotest in patients with alcoholic liver disease and to compare their diagnostic performances., Patients and Methods: We prospectively included 200 consecutive patients (159 men and 41 women; mean age: 51±0.7 years).The PGAA index was calculated by combining the results of four laboratory tests (prothrombin time, γ-glutamyl transpeptidase, apolipoprotein A1, and α-2-macroglobulin) scored on a 0-4 scale. The Fibrotest score was computed using the Biopredictive website. The overall diagnostic performances of scores were evaluated in terms of the area under the receiver operating characteristic (AUROC) curve. The Obuchowski measure was assessed taking into account the distribution of fibrosis stages observed in the cohort., Results: For predicting F≥2 fibrosis stage, the AUROC curves of PGAA and Fibrotest were 0.83±0.03 and 0.80±0.03, respectively. For predicting F4 fibrosis stage, the AUROC curves of PGAA and Fibrotest were 0.87±0.03 and 0.86±0.03. There was no difference between the AUROC curves of PGAA and Fibrotest. The Obuchowski measure was 0.92±0.01 for PGAA and Fibrotest. For a value of 10, PGAA had 98% specificity and 97% positive predictive value for the detection of F≥2 fibrosis stage and 80% sensitivity and 92% negative predictive value for F4 stage fibrosis., Conclusion: We confirm the comparable diagnostic values of Fibrotest and PGAA. When Fibrotest use is constrained by an increase in unconjugated bilirubin or is not financially viable, PGAA may be an alternative.

Background: Nasal mucus is the first-line defense barrier against (aero-) allergens. However, its proteome and function have not been clearly investigated., Objective: The role of nasal mucus in the pathophysiology of allergic rhinitis was investigated by analyzing its proteome in patients with allergic rhinitis (n = 29) and healthy control subjects (n = 29)., Methods: Nasal mucus was collected with a suction device, tryptically digested, and analyzed by using liquid chromatography-tandem mass spectrometry. Proteins were identified by searching the SwissProt database and annotated by collecting gene ontology data from databases and existing literature. Gene enrichment analysis was performed by using Cytoscape/BINGO software tools. Proteins were quantified with spectral counting, and selected proteins were confirmed by means of Western blotting., Results: In total, 267 proteins were identified, with 20 (7.5%) found exclusively in patients with allergic rhinitis and 25 (9.5%) found exclusively in healthy control subjects. Five proteins were found to be significantly upregulated in patients with allergic rhinitis (apolipoprotein A-2 [APOA2], 9.7-fold; α2-macroglobulin [A2M], 4.5-fold; apolipoprotein A-1 [APOA1], 3.2-fold; α1-antitrypsin [SERPINA1], 2.5-fold; and complement C3 [C3], 2.3-fold) and 5 were found to be downregulated (antileukoproteinase [SLPI], 0.6-fold; WAP 4-disulfide core domain protein [WFDC2], 0.5-fold; haptoglobin [HP], 0.7-fold; IgJ chain [IGJ], 0.7-fold; and Ig hc V-III region BRO, 0.8-fold) compared with levels seen in healthy control subjects., Conclusion: The allergic rhinitis mucus proteome shows an enhanced immune response in which apolipoproteins might play an important role. Furthermore, an imbalance between cysteine proteases and antiproteases could be seen, which negatively affects epithelial integrity on exposure to pollen protease activity. This reflects the important role of mucus as the first-line defense barrier against allergens., (Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Objectives: The aim of this study was to independently validate proteomic biomarkers previously reported to be differentially expressed in women with Polycystic Ovary Syndrome (PCOS) compared with controls. This study focused on plasma proteomic biomarkers., Methods: This was a cross-sectional study at the University of Nottingham, in which samples from 30 PCOS and 30 control women were analysed by Western blotting., Results: Mean abundance ratios from Western blots of plasma total haptoglobin and haptoglobin beta proteins were 1.25 (CI 1.11-1.4) and 1.24 (CI 1.04-1.44). The mean abundance ratio from the blots of alpha-2 macroglobulin was however 1.05 (CI, 1-1.1). The mean PCOS/control BMI ratio was 1.18 (CI 1.17-1.20). There was no correlation between PCOS/control BMI ratio and alpha-2 macroglobulin, total haptoglobin and haptoglobin beta abundance ratios. There was also no correlation between PCOS/control insulin ratio and alpha-2 macroglobulin, total haptoglobin and haptoglobin beta abundance ratios., Conclusions: Total haptoglobin and haptoglobin beta chain protein abundance was found to be elevated in women with PCOS compared with controls. We were unable to confirm decreased alpha-2 macroglobulin levels as reported in a previous study. Independent validation studies are required to validate early promising proteomic biomarkers in PCOS.

Adipocyte differentiation is a complex developmental process forming adipocytes from various precursor cells. The murine 3T3-L1 preadipocyte cell line has been most frequently used in the studies of adipocyte differentiation. Differentiation of 3T3-L1 preadipocytes includes a medium containing fetal bovine serum (FBS) with hormonal induction. In this study, we observed that differentiation medium containing adult bovine serum (ABS) instead of FBS did not support differentiation of preadipocytes. Impaired adipocyte differentiation was due to the presence of a serum protein factor in ABS that suppresses differentiation of preadipocytes. Using a proteomic analysis, alpha-2-macroglobulin and paraoxonase/arylesterase 1, which were previously shown to suppress differentiation of preadipocytes, were identified as anti-adipogenic proteins. Although their functional mechanisms have not yet been elucidated, the anti-adipogenic effects of these proteins are discussed.

A novel form of ovomacroglobulin/ovostatin (OVOS2) predicted from EST data was previously identified in the chicken ovarian cancer model using a mass spectrometry-based shotgun label-free proteomics strategy. The quantitative label-free data from plasma showed a significant increase over time with the spontaneous onset and progression of ovarian cancer making it a potential protein biomarker for further study. Two other proteins of interest identified from this initial study included vitellogenin-1 (Vit-1), a lipid-transport protein tied to egg production, and transthyretin (TTR), a retinol binding transport protein currently used in the clinical management of ovarian cancer. A multiplexed protein cleavage isotope dilution mass spectrometry (PC-IDMS) assay was developed to quantify OVOS2, Vit-1, and TTR by selected reaction monitoring (SRM). A total of 6 stable isotope labeled (SIL) peptide standards were used in the assay with three tryptic peptides from OVOS2, one for Vit-1, and two for TTR. The assay was developed for use with un-depleted raw plasma combined with the filter assisted sample preparation (FASP) method and its use was also demonstrated for matched ovary tissue samples. The PC-IDMS data for the two TTR peptides did not correlate with each other with more than a 10-fold difference in concentration for all 5 time points measured. The PC-IDMS data from the longitudinal plasma samples correlated well for OVOS2 and Vit-1 whereas TTR was inconclusive. Interestingly, the absolute amount for one of the OVOS2 SIL peptides was 2-fold less compared with the other two SIL peptides. These data illustrate the successes and challenges of qualifying quantitative levels of proteins from an in-gel digestion sample preparation followed by LC-MS/MS (GeLC) label-free discovery-based approach to a targeted SRM-based quantitative assay in plasma and tissues., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Beta-2 microglobulin (B2M) and alpha-2 macroglobulin (A2M) play key roles in the immune system. The aim of this study was to compare B2M and A2M levels in patients with different periodontal diseases., Methods: Eighty patients (20 periodontally healthy, 20 with gingivitis, 20 with chronic periodontitis and 20 with generalized aggressive periodontitis) were enrolled in the study. The analysis of B2M and A2M was performed on gingival crevicular fluid (GCF) using an enzyme-linked immunosorbent assay in GCF., Results: The total levels of B2M and A2M were statistically lower in the periodontally healthy group than in the other groups (p < 0.05) and significantly higher in the generalized aggressive periodontitis group compared to the other groups (p < 0.05)., Conclusions: B2M and A2M play key roles in the balance between periodontal health and disease. It is proposed that tissues release B2M and A2M to stop inflammation and inhibit the proliferation of microorganisms and this may be the reason for the high levels of B2M and A2M in the generalized aggressive periodontitis and chronic periodontitis groups. B2M and A2M are assumed to be user-friendly and cost-effective markers for periodontal disease to identify asymptomatic diseases., (© 2013 Australian Dental Association.)

Objective: Despite the number of potential biomarker proteins for diabetes, very few of them have proven useful as clinically beneficial indicators, because of the technical difficulties associated with their identification among highly abundant serum proteins. We attempted to identify a protein with distinguishable expression in human diabetes., Methods: We applied a highly efficient strategy for the purification of endogenous low abundance proteins from diabetic and non-diabetic serum samples. Extracted sera were fractionated by SDS-PAGE and protein bands were isolated and analyzed by mass spectrometry using an ion-trap mass spectrometer. The identities of the proteins were confirmed by western blotting and the serum levels evaluated., Results: A significantly upregulated protein in diabetic patients was identified as monomeric α2-macroglobulin. Its tetramer, another dominant circulating molecular form, was only marginally increased in diabetes., Conclusion: Serum monomeric α2-macroglobulin is highly expressed in many diabetic subjects. It is identical to the human 'cardiac isoform of α2-macroglobulin' described in the literature, a well-known acute phase serum biomarker protein mechanistically involved in cardiac and atherosclerotic diseases., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Background: Serum hyaluronic acid and biochemical models which require hyaluronic acid analysis are commonly used as predictors of liver fibrosis in patients with chronic liver disease, however biological variation data for hyaluronic acid are deficient., Methods: Four serial serum samples were obtained at weekly intervals from healthy volunteers and patients with chronic hepatitis B, chronic hepatitis C and non- alcoholic fatty liver disease (NAFLD; 20 in each group). The within-individual week-to-week variation (CVI) and reference change values for hyaluronic acid, α₂-macroglobulin and Hepascore were obtained. Hepascore is calculated from hyaluronic acid, α2-macroglobulin, bilirubin and γ-glutamyltransferase activity., Results: Hyaluronic acid displayed large within-individual variation, the CVI values were 62% in healthy subjects, 38% in hepatitis C, 37% in hepatitis B and 36% in NAFLD patients. Hepascore CVIs were 43% in healthy subjects, 24% in hepatitis C, 28% in hepatitis B and 39% in NAFLD patients. α₂-Macroglobulin was much less variable with CVIs ranging from 4.4% to 7.6%. Bland-Altman plots of week-to-week variations showed rates of significant disagreement for samples collected in any 2 successive weeks varied from 5% in NAFLD patients to 8.3% in healthy subjects., Conclusions: When using non-fasting serum samples, hyaluronic acid and to a lesser extent, the Hepascore model display large within-individual variations in both health and chronic liver disease. This information is critical for interpreting the significance of both single measurements and changes in serial measurements.

Background: Due to the lack of cutoff values validated for specific liver diseases, the purpose of this study was to set up specific magnetic resonance elastography (MRE) cutoff values for asymptomatic liver fibrosis in alcoholic patients., Methods: Ninety patients underwent 3 clinical exams. The liver stiffness was measured locally with the Fibroscan, and globally through cartographies of shear modulus generated with MRE. The Fibroscan method was chosen as the gold standard to classify the fibrosis. The liver score was also obtained with the Fibrometer A, and the diagnostic performance of the methods was analyzed with receiver-operating characteristic (ROC) curves and cutoff values were calculated., Results: Spearman correlation and area under the ROC curve revealed that MRE is a better diagnostic method than the Fibrometer A, to identify various levels of fibrosis. The results showed that the Fibrometer A was adapted for severe fibrosis. The MRE cutoff values are F1:2.20 kPa, F2:2.57 kPa, F3:3.31 kPa, and F4:4 kPa and were not influenced by the glutamic oxaloacetic transaminase level. By using the ultrasound cutoff values attributed for alcoholism, 66% of patients had a similar liver fibrosis diagnosis as the MRE cutoffs. However, both imaging techniques did not provide the same distribution for minor fibrosis., Conclusions: None of the imaging techniques (Fibroscan, MRE) could replace the gold standard of the biopsy. However, due to the risk and the unnecessary procedure for the present recruited alcoholic patients, the Fibroscan method was chosen as the reference. Since MRE is currently being used as a clinical exam, the present MRE cutoffs could aid clinicians with their diagnosis of liver fibrosis for alcoholism disease., (Copyright © 2012 by the Research Society on Alcoholism.)

Tuberculosis (TB) in children is not only more likely to cause more severe disease than that seen in adults, it is also more likely to be extrapulmonary. Moreover, pediatric TB is very difficult to diagnose and suffers from a lack of understanding of host biomarkers for monitoring the progression of disease. Hence, we sought to identify the expression patterns of a variety of biomarkers in the plasma of children with pulmonary TB (PTB) and extrapulmonary TB (ETB), as well as in healthy control (HC) children. Thus, we examined a variety of circulating markers reflecting tissue inflammation, oxidative stress, innate immune activation, fibrosis, and the cytokine response. Children with active TB, compared to HC children, showed markedly elevated plasma levels of matrix metalloproteinases and their endogenous inhibitors. In addition, children with active TB had significantly elevated levels of C-reactive protein, α-2 macroglobulin, and haptoglobin, as well as hemoxygenase 1. Markers of innate immune activation (lipopolysaccharide [LPS] and lipopolysaccharide-binding protein [LBP]) were significantly lower in ETB than in PTB children. Although there were no significant differences between the two groups in their levels of cytokines (type 1 [gamma interferon (IFN-γ), tumor necrosis factor α (TNF-α), interleukin 2 (IL-2), and IL-12], type 2 [IL-4, IL-5, IL-13, and IL-33], and most type 17 [IL-17A, IL-22, IL-1β, and IL-6] and type 1 interferons [IFN-α and IFN-β]) or most of the cytokines associated with immune modulation (IL-10 and IL-20), pediatric TB was associated with elevated plasma transforming growth factor β (TGF-β), IL-21, and IL-23 levels. Thus, pediatric TB is characterized by elevated levels of a variety of biomarkers at homeostasis, suggesting that these responses may play a crucial role in disease pathogenesis.

Background: Ischemic preconditioning (IPC) has been shown to protect the liver against ischemia-reperfusion (I/R) injuries. However, ischemic post-conditioning has received little attention. The aim of the present study was to quantify and compare the hepato-protective properties of IPC and IPO, for the first time, using unbiased design-based stereological methods., Methods: We divided 67 rats into four groups: sham, liver ischemia (LI), IPC, and IPO. Rats were subjected to 60 min LI, followed by 4- or 24-h reperfusion. We performed quantification of (NVR) and apoptotic cell profile number., Results: We observed no significant differences in NVR between ischemic groups after 4 h. After 24-h reperfusion, NVR had increased to 70% in the LI group, compared with 51% (P = 0.02) and 49% (P = 0.01) in the IPC and IPO groups, respectively. After 4-h reperfusion, the apoptotic cell number was significantly higher in all ischemic groups than in the sham group; we detected no difference between ischemic groups. After 24-h reperfusion, we detected a significantly lower number of apoptotic cell profiles in the IPC group than in the LI group (P = 0.02). The mean number of apoptotic cell profiles decreased insignificantly in the IPO group (P = 0.06). Liver parameters were at all time comparable between groups., Conclusions: After I/R, IPC and IPO reduce the degree of hepatocellular injury. Both methods are equally efficient at preventing hepatocellular necrosis. Furthermore, apoptosis is significantly lower after IPC., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Noninvasive methods for liver fibrosis evaluation in chronic liver diseases have been recently developed, i.e. transient elastography (Fibroscan™) and blood tests (Fibrometer®, Fibrotest®, and Hepascore®). In this study, we aimed to design a new score in chronic hepatitis C (CHC) by selecting blood markers in a large panel and we compared its diagnostic performance with those of other noninvasive methods., Methods: Sixteen blood tests were performed in 306 untreated CHC patients included in a multicenter prospective study (ANRS HC EP 23 Fibrostar) using METAVIR histological fibrosis stage as reference. The new score was constructed by non linear regression using the most accurate biomarkers., Results: Five markers (alpha-2-macroglobulin, apolipoprotein-A1, AST, collagen IV and osteoprotegerin) were included in the new function called Coopscore©. Using the Obuchowski Index, Coopscore© shows higher diagnostic performances than for Fibrometer®, Fibrotest®, Hepascore® and Fibroscan™ in CHC. Association between Fibroscan™ and Coopscore© might avoid 68% of liver biopsies for the diagnosis of significant fibrosis., Conclusion: Coopscore© provides higher accuracy than other noninvasive methods for the diagnosis of liver fibrosis in CHC. The association of Coopscore© with Fibroscan™ increases its predictive value., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: The main causes of liver fibrosis in transfusion-dependent thalassemia major are hepatitis C virus (HCV) infection and hepatic iron overload. The study aimed to assess liver fibrosis in Egyptian adolescents and young adult poly-transfused beta thalassemia patients infected with HCV using liver FibroScan in relation to iron overload and Liver iron concentration (LIC)., Material and Methods: Fifty-one regularly transfused beta thalassemia patients above 12 years old were subjected to measurement of serum alanine transaminase (ALT), serum ferritin (SF), HCV (antibody and RNA), LIC assessed by hepatic R2* and transient elastography (TE) (FibroScan). FibroTest and liver biopsy were done to 25 patients., Results: Eighty two% of studied thalassemia patients were HCV antibody positive; 21(49%) of them were viremic (HCV RNA positive); median LIC was 12 mg/gm dry weight. There were strong positive correlation between the degree of liver stiffness and Ishak fibrosis score assessed in liver biopsy specimens (P = 0.002) and between FibroScan and FibroTest results (P < 0.001). Patients with HCV viremia showed significantly higher ALT, γ-glutamyl transpeptidase (GGT), SF, LIC and increased liver stiffness compared to patients with no viremia (P = 0.0001, 0.001, 0.012, 0.006 and 0.001) respectively. Liver cirrhosis (TE values > 12.5kPa) was encountered in 23.5% and variable degrees of liver fibrosis (TE values > 6-12.5 kPa) in 35% of studied thalassemic patients., Conclusion: Young beta thalassemia patients with active hepatitis C infection may have hepatic cirrhosis or fibrosis at young age when accompanied with hepatic siderosis. Non invasive Liver FibroScan and Fibro-Test were reliable methods to assess liver fibrosis in young thalassemic-patients.

Background: There is an ongoing controversy on the benefits of treatment protocols, including dental lasers and photodynamic therapy (PDT). The purpose of this study is to compare the local biologic effects of PDT, diode soft laser (DSL) therapy, and conventional deep scaling and root planing (SRP) in residual pockets., Methods: Thirty-two individuals were included based on a history of previous treatment for periodontitis and the persistence of sites with probing depths >4 mm and bleeding on probing. Residual pockets were debrided with an ultrasonic device and then randomly assigned either to PDT, DSL, or SRP. Gingival crevicular fluid was collected before treatment, after 14 days, and at 2 and 6 months. Levels of 13 cytokines and nine acute-phase proteins were measured using a bead-based multiplexing analysis system., Results: Treatment with PDT, DSL, or SRP led to significant changes in several cytokines and acute-phase proteins: Compared with baseline, levels of interleukin-17, basic fibroblast growth factor, granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor, and macrophage inflammatory protein 1-α were lower 14 days and 2 months after treatment. Except for granulocyte colony-stimulating factor, these differences remained significant throughout the observation period. The levels of five acute-phase proteins (α-2 macroglobulin, haptoglobin, serum amyloid P, procalcitonin, and tissue plasminogen activator) were significantly higher at 6 months than at baseline. No significant differences were observed among the three treatment modalities at any time point for any biochemical parameter., Conclusions: Levels of several cytokines and acute-phase proteins significantly changed after treatment regardless of treatment modality. There was no evidence for a specific DSL- or PDT-enhanced expression of inflammatory mediators.

Viral diseases restrict the development of the world shrimp industry and there are few studies on cell response to the presence of viral infections. We performed immunohistochemistry assays to characterize hemocytes subpopulations involved in the immune process occurring in the LO of Litopenaeus vannamei shrimp. Tissue sections of animals that increased their LO spheroids and hemocytes infiltration after WSSV induced infection, were used. Three MABs namely, 40E10 (recognizing small granule hemocytes), 40E2 (recognizing large granule hemocytes), and 41B12, which recognize α(2)-macroglobulin were used. Additionally one polyclonal antibody was used against the penaeidins antimicrobial peptides, and to detect WSSV a commercial immunohistochemistry kit (DiagXotics) was used. Numerous small granule hemocytes were detected in the stromal matrix of LO tubules, whereas large granule hemocytes were less numerous and located mainly in hemal sinuses. The exocytosis of two molecules, which have been related to the phagocytosis process, i.e. penaeidins, and α(2)-macroglobulin, was detected in the external stromal matrix and the outer tubule walls. α(2) -macroglobulin inhibits phenoloxidase activity and its strong release in LO tissue may explain the absence of melanization in the immune processes occurring in it. The immunolabeling of vesicles within the LO spheroids with MABs 41B12 40E10 and antipenaedin antibody suggests that LOS are formed by phagocytic cells derived from small granule and hyaline hemocytes, with a possible role of peneidins and α(2)-macroglobulin acting as opsonines., (© 2012 The Societies and Blackwell Publishing Asia Pty Ltd.)

We investigate the efficacy of poly D,L-lactide-co-glycolic acid (PLGA)-encapsulated vaccine on innate and adaptive immune response in kelp grouper (Epinephelus bruneus) against Uronema marinum at weeks 1, 2, and 4. The respiratory burst (RB) activity, complement activity, and α2-macroglobulin were significantly enhanced in fish immunization with vaccine on week 4 whereas vaccine and PLGA-encapsulated vaccine from weeks 1 to 4. The serum lysozyme activity, antiprotease activity, and antibody level were significantly enhanced in fish immunized with vaccine and PLGA-encapsulated vaccine on weeks 2 and 4. The cumulative mortality was low in PLGA-encapsulated vaccine with 20% whereas high in PLGA and vaccine with 40% and 30%. The results from the present study suggest that PLGA-encapsulated vaccine is useful for further design of immunoprophylatic nano formulation against scuticociliatosis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Introduction: Alpha-2-macroglobulin (α(2)M) is a broad specificity protease inhibitor which impacts several hemostatic pathways. Selective detection of various α(2)M complexes may be useful to define markers for the status of different hemostatic components. We present proof of principle for a novel assay to quantitatively measure α(2)M in complex with a variety of hemostatic factors., Materials and Methods: The assay makes use of the fact that α(2)M entraps proteases within a molecular "cage", leaving them inaccessible to macromolecular substrates while retaining functionality against small synthetic substrates. Wells coated with anti-α(2)M antibodies were used to isolate the complexes from buffer or plasma, followed by detection of specific proteases with chromogenic substrates. Macromolecular inhibitors were added to eliminate signal from any unbound proteases., Results: Calibration curves constructed with purified protease-α(2)M complexes were sigmoidal in nature, as is typical with immuno-assays. The specificity of signal production was confirmed with inhibitors that target either free protease, or both free and α(2)M-bound protease. The detection range of the assay was dependent on the protease being measured, and the surrounding matrix. Interference in detection of complexes in plasma was found to be caused, in part, by free α(2)M. Thrombin-α(2)M complexes were quantified in adult and newborn plasma following induction of thrombin generation and found to be significantly higher in adults, likely due to higher prothrombin levels., Conclusions: This assay provides a versatile platform method for quantification of multiple protease-α(2)M complexes. It may prove useful for mechanistic in vitro studies of hemostatic pathways, and potentially for clinical applications., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Patients with diabetes are at risk for nonalcoholic fatty liver disease leading to cirrhosis. Existing guidelines do not advocate screening for liver related complications amongst persons with diabetes., Aim: The aim of this prospective study was to identify patients with severe liver fibrosis amongst patients hospitalized for their diabetes, using non-invasive methods, and to evaluate factors associated with severe fibrosis., Methods: Consecutive patients with type 1 or 2 diabetes had clinical, biological parameters and liver fibrosis evaluation. Severe fibrosis was predicted when FibroTest was >0.59 or liver stiffness >8.7 kPa., Results: A total of 277 patients were evaluated (type 1 diabetes 52%). The prevalence of severe fibrosis was 15.5%. By univariate analysis, factors associated with severe fibrosis were age, type 2 diabetes, body mass index, metabolic syndrome, previous cardiovascular events, no retinopathy, past history of foot ulcer, and elevated alanine aminotransferase. By multivariate analysis, factors associated with severe fibrosis were age >50 years, type 2 diabetes, no retinopathy, and past history of foot ulcer., Conclusion: This study showed an elevated prevalence of severe fibrosis in hospitalized diabetic patients, especially patients aged 50 years or older with type 2 diabetes, or with a past history of foot ulcer., (Copyright © 2011 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Purpose: To evaluate the utility of an enhanced biomarker discovery approach in order to identify potential biomarkers relevant to ovarian cancer detection., Experimental Design: We combined immuno-depletion, liquid-phase IEF, 1D-DIGE, MALDI-TOF/MS and LC-MS/MS to identify differentially expressed proteins in the plasma of symptomatic ovarian cancer patients, stratified by stage, compared to samples obtained from normal subjects., Results: We demonstrate that this approach is a practical alternative to traditional 2D gel techniques and that it has some advantages, most notably increased protein capacity. Proteins were identified in all 76 bands excised from the gels in this project and confirmed the cancer-associated expression of several well-established biomarkers of ovarian cancer. These included C-reactive protein (CRP), haptoglobin, alpha-2 macroglobulin and A1A2. We also identified new ovarian cancer candidate biomarkers, Protein S100-A9 (S100A9) and multimerin-2. The cancer-associated differential expression of CRP and S100A9 was further confirmed by Western blot and ELISA., Conclusions: The methods developed in this study allow for the increased loading of plasma proteins into the analytical stream when compared to traditional 2D-DIGE. This increased protein identification sensitivity allowed us to identify new putative ovarian cancer biomarkers., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: To evaluate the predictive value of a set of laboratory markers for the assessment of liver fibrosis in chronic viral hepatitis patients., Study Design: Cross-sectional study., Place and Duration of Study: Baqai Medical University, Combined Military Hospital, Malir, Karachi, from November 2006 to May 2008., Methodology: Twenty laboratory parameters were measured in 100 treatment-naïve chronic viral hepatitis patients who also had liver biopsy performed. Descriptive statistics, areas under the ROC's curves, and multivariate logistic regression analysis identified a fibrosis panel, a set of five most useful markers, for the assessment of stages of fibrosis, stage 0 to stage 4. The fibrosis index, FibroScore, consisted of bilirubin, Gamma glutamyl transferase, Hyaluronic acid, alpha 2 macroglobulin, and platelets evaluation., Results: A score of ≥ 0.5 predicted stages 2, 3 and 4, with a sensitivity of 82%, and specificity of 92%. A score ≥ 0.5 for stages 3 and 4 had a sensitivity of 85%, and specificity of 89%. At a score of > 0.80, for stages 3 and 4, the sensitivity was 70%, specificity was 97%, and PPV 87% (there was ≥ 85% possibility of presence of stage 3 or 4). A score of ≤ 0.20 predicted the absence of stages 2, 3, and 4 with a sensitivity of 91%, specificity of 86%, and NPV of 96%. Scores from 0.00 to 0.10 almost certainly ruled out the presence of stages 2-4 (NPV=98%). The areas under the ROC curve were: 0.808 for stage 2; 0.938 for stage 3; and 0.959 for stage 4., Conclusion: A combination of 5 markers is very useful in predicting various stages of liver fibrosis, and is helpful in the non-invasive assessment of liver fibrosis in chronic viral hepatitis patients.

A method in which a permanent magnet is introduced onto polydiacetylene (PDA) vesicle chips is introduced for enhancement of the fluorescence of PDA vesicles. This strategy can be applied to general antibody-based PDA vesicle chips to detect clinically important biomarkers for disease diagnosis., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction: Osteoarthritis (OA) is a degenerative disease characterized by cartilage breakdown in the synovial joints. The presence of low-grade inflammation in OA joints is receiving increasing attention, with synovitis shown to be present even in the early stages of the disease. How the synovial inflammation arises is unclear, but proteins in the synovial fluid of affected joints could conceivably contribute. We therefore surveyed the proteins present in OA synovial fluid and assessed their immunostimulatory properties., Methods: We used mass spectrometry to survey the proteins present in the synovial fluid of patients with knee OA. We used a multiplex bead-based immunoassay to measure levels of inflammatory cytokines in serum and synovial fluid from patients with knee OA and from patients with rheumatoid arthritis (RA), as well as in sera from healthy individuals. Significant differences in cytokine levels between groups were determined by significance analysis of microarrays, and relations were determined by unsupervised hierarchic clustering. To assess the immunostimulatory properties of a subset of the identified proteins, we tested the proteins' ability to induce the production of inflammatory cytokines by macrophages. For proteins found to be stimulatory, the macrophage stimulation assays were repeated by using Toll-like receptor 4 (TLR4)-deficient macrophages., Results: We identified 108 proteins in OA synovial fluid, including plasma proteins, serine protease inhibitors, proteins indicative of cartilage turnover, and proteins involved in inflammation and immunity. Multiplex cytokine analysis revealed that levels of several inflammatory cytokines were significantly higher in OA sera than in normal sera, and levels of inflammatory cytokines in synovial fluid and serum were, as expected, higher in RA samples than in OA samples. As much as 36% of the proteins identified in OA synovial fluid were plasma proteins. Testing a subset of these plasma proteins in macrophage stimulation assays, we found that Gc-globulin, α1-microglobulin, and α2-macroglobulin can signal via TLR4 to induce macrophage production of inflammatory cytokines implicated in OA., Conclusions: Our findings suggest that plasma proteins present in OA synovial fluid, whether through exudation from plasma or production by synovial tissues, could contribute to low-grade inflammation in OA by functioning as so-called damage-associated molecular patterns in the synovial joint.