Plasma Of Healthy Controls Research Articles

Catalase activity in blood fractions of patients with sporadic ALS

BackgroundOxidative stress may be a key element in pathogenesis of sporadic amyotrophic lateral sclerosis (sALS). Several studies proved that markers of peroxidation of lipids, proteins or nucleic acids are increased in postmortem tissue of sALS patients. However, much less is known about enzymatic antioxidant defense mechanism in sALS. ObjectivesThe aim of the study was to assess catalase (CAT) activity that is implicated in the defense against oxidative stress, in three blood fractions, i.e. erythrocytes, plasma and serum of sALS patients and healthy controls. MethodsAltogether 46 sALS patients and 54 controls were enrolled in the study. CAT activity was estimated using a commercially available assay kit. ResultsCAT activity in erythrocytes of sALS patients was significantly decreased compared to neurologically healthy controls (p=0.04), whereas CAT activity in plasma and serum was similar in both studied groups. ConclusionsCAT activity in erythrocytes, in contrast to other blood fractions is reduced in sALS cases as compared to controls, which may indirectly indicate that antioxidant defense system in erythrocytes is involved in pathogenesis of sALS.

Soluble TNF receptors are produced at sites of inflammation and are inversely associated with self-reported symptoms (WOMAC) in knee osteoarthritis.

The aim of the study was to analyze the concentrations of sTNFR1 and sTNFR2 in both plasma and synovial fluid of patients with primary knee osteoarthritis (OA) and to determine their relationship to self-reported pain, stiffness and physical function. Twenty-seven patients with knee OA and 19 healthy subjects were enrolled in the study. The Western Ontario and McMaster University Osteoarthritis Index questionnaire was used to evaluate self-reported physical function, pain and stiffness. The sTNFR1 and sTNFR2 levels in the plasma and synovial fluid were measured by enzyme-linked immunosorbent assay. The sTNFR1 levels in synovial fluid of OA patients (2,587 ± 66.12 pg/mL) were 2.5-fold higher than in corresponding blood samples and were 1.5-fold higher than in the plasma of healthy controls. The plasma sTNFR2 levels in the patients with knee OA were lower than in healthy controls (2,249 ± 126.3 vs. 2,700 ± 126.3 pg/mL, p < 0.05), and sTNFR2 levels in synovial fluid of knee OA patients (2,021 ± 107.0 pg/mL) were lower than in the plasma of healthy controls. Synovial fluid sTNFR1 levels were negatively correlated with pain and physical function self-reported (r s - 0.6785, p < 0.0001 and r s - 0.4194, p = 0.03, respectively). Synovial fluid sTNFR2 levels were negatively correlated with pain and joint stiffness (r s - 0.5433, p = 0.01 and r s - 0.4249, p = 0.02, respectively). The findings of this study demonstrated the presence of soluble receptors for TNF-alpha, particularly sTNFR1, in the synovial fluid of patients with primary knee OA and the relationship of these receptors with clinical parameters.

Involvement of BDNF in knee osteoarthritis: the relationship with inflammation and clinical parameters

The aim of this study was to analyze the levels of brain-derived neurotrophic factor (BDNF) in both the plasma and synovial fluid of patients with primary knee osteoarthritis compared with control individuals and to investigate the relationship between BDNF levels and self-reported pain. Twenty-seven patients with knee osteoarthritis (OA) and 19 healthy subjects were enrolled in the study. Anteroposterior knee radiographs were taken to determine the disease severity of the affected knee. Radiographic grading of OA in the knee was performed using the Kellgren-Lawrence criteria. The BDNF levels in the plasma and synovial fluid were measured by enzyme-linked immunosorbent assay. The mean plasma BDNF levels of the knee OA patients were significantly higher than that of the healthy controls (2,378 ± 1,067.2 vs. 1,756 ± 804.3 pg/mL, p < 0.05). BDNF levels in the synovial fluid of OA patients (358.9 ± 178.4 pg/mL) were sixfold lower than in corresponding blood samples (p < 0.0001) and fourfold lower than in the plasma of healthy controls (p < 0.0001). Subsequent analyses showed that the plasma BDNF levels significantly correlated with self-reported pain (Western Ontario and McMaster Universities Osteoarthritis Index) (r s = 0.39, p = 0.04). Furthermore, no correlation was found between the plasma and synovial fluid BDNF concentrations and knee OA severity. The findings of this study suggest that systemic BDNF levels are most likely associated with the mechanism of joint pain in knee OA in the acute stage of joint inflammatory process. Further studies are necessary to address the functional role of BDNF in the modulation of pain to establish new therapeutic implications.

Analysis Of Hemostatic Characteristics using Thromboelastometry in Adults With Sickle Cell Disease and Controls

Background Vaso-occlusive phenomena and hemolysis are the clinical hallmarks of sickle cell disease (SCD). In addition, pain crisis was identified as the initial clinical manifestation in 61.9% of sickle cell patients who died shortly after hospital admission from thromboembolism and micro vascular thrombi. Knowing that the vaso-occlusive events may be related to activation of the hemostatic system, and that thromboelastometry (TEM) assesses the functionality of this system from a global standpoint, it will be challenging to characterize the findings in patients with SCD as a way to differentiate their clinical phenotype upon presentation, and to predict the impact of these manifestations on their prognosis, mortality and morbidity. Objective To characterize the findings of TEM in patients with SCD during periods of steady state and acute illness, to compare these results with those of healthy controls and sickle cell trait (SCT), to compare the findings in whole blood (WB) to plasma (PL) for each category, in a way to analyze the findings in plasma and their applicability in clinical practice as well as to delineate the contribution of the cellular component in whole blood samples. Design In a cross-sectional study, we obtained TEM and other hemostatic data on 24 adult patients with SCD (16 in steady state and 8 in acute illness); and 13 race and age matched healthy controls (6 with sickle cell trait (SCT) and 7 with no trait). We specifically studied coagulation time (CT) as a function of coagulation factors; clot firmness time (CFT) and alpha angle(α) assessing platelet and fibrinogen function; and maximum clot firmness (MCF) evaluating the mechanical clot quality (plt, fibrinogen and factorXIII) and finally thrombodynamic potential index (TPI) as a function of patient’s global coagulation. Results Overall, patients with SCD had higher TPI in WB (p=0.23;=0.25) and lower TPI in PL (p&lt;0.0001;=0.47) when compared to controls and SCT respectively. Also, patients with SCD had lower CT (p=0.02), lower CFT (p&lt;0.0001) higher MCF; α and TPI (p&lt;0.0001; =0.051; &lt;0.0001) in whole blood compared to plasma. Sickle cell trait patients had lower CT, CFT, alpha with higher MCF in WB compared to PL. While healthy controls had higher CT; MCF and TPI (p=0.01; &lt;0.0001; &lt;0.0001) and lower CFT, α (p=0.16; &lt;0.0001) in WB compared to PL. Conclusion Whole blood of SCD patients seems to be hypercoagulable in comparison to WB of controls and SCT. While the plasma of SCD patients was significantly hypocoagulable when compared to PL of healthy controls. Overall, TEG profiles of WB were different than PL. This was more obvious in SCD patients reinforcing the contribution of the cellular component to the pathophysiology of this disease and the possible compensatory hypocoagulable status of the plasma in these patients. Further study of larger and more homogeneous patient groups, is required to adequately assess the clinical utility of TEM in patients with sickle cell disease. Disclosures: Kruse-Jarres: Baxter Healthcare: Consultancy; Bayer HealthCare: Consultancy; Biogen IDEC: Consultancy; Grifols: Consultancy; Kedrion: Consultancy; Novo Nordisk: Consultancy.

EVALUATION OF THE PLASMA MICRO RNA EXPRESSION LEVELS IN SECONDARY HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS

BackgroundHemophagocytic lymphohistiocytosis (HLH) is a life threatening hyper inflammatory disease. Micro RNAs (miRNA) are about 22 nucleotide-long, small RNAs encoded with genes, and they have regulatory functions in immune response.ObjectiveTo determine the miRNA expression levels of 11 secondary HLH patients, we evaluated the associations of miRNA levels with pathogenesis, clinical presentation, and prognosis of the disease.Patients and MethodsPatients who were diagnosed with secondary HLH from January 2011 to December 2012 were included in this study. We profiled the expressions of 379 miRNAs in plasma of both HLH patients and healthy controls. Patients were evaluated regarding with age, clinical findings, miRNA expresions, laboratory data, treatment, and prognosis, by using descriptive statistics.ResultsA total of 11 secondary HLH patients and 11 healthy children were included in this study. miR-205-5p was expressed in all case and controls and expression level of miR-205-5p was found 6.21 fold higher than control group (p=0.01). We detected the second highest expression percent in miR-194-5p with 81% of cases and controls. Expression level of miR-194-5p was found to have 163 fold higher than controls (p= 0.009). miR-30c-5p showed 77% expression percent in cases and controls together. The expression level of this miRNA was detected 9 fold decreased in HLH patients compared to healthy children (p= 0.031).ConclusionWe showed that miR-205-5p, miR-194-5p and miR-30c-5p could be useful plasma biomarkers for HLH. Further research is needed in larger and homogenous study groups, especially for these miRNAs as biomarkers for HLH.

Neuroactive Steroid Levels are Modified in Cerebrospinal Fluid and Plasma of Post-Finasteride Patients Showing Persistent Sexual Side Effects and Anxious/Depressive Symptomatology

Observations performed in a subset of subjects treated with finasteride (an inhibitor of the enzyme 5α-reductase) for male pattern hair loss seem to indicate that sexual dysfunction as well as anxious/depressive symptomatology may occur at the end of the treatment and continue after discontinuation. A possible hypothesis to explain depression symptoms after finasteride treatment might be impairment in the levels of neuroactive steroids. Therefore, neuroactive steroid levels were evaluated in paired plasma and cerebrospinal fluid samples obtained from male patients who received finasteride for the treatment of androgenic alopecia and who, after drug discontinuation, still show long-term sexual side effects as well as anxious/depressive symptomatology. The levels of neuroactive steroids were evaluated by liquid chromatography-tandem mass spectrometry in three postfinasteride patients and compared to those of five healthy controls. Neuroactive steroid levels in plasma and cerebrospinal fluid of postfinasteride patients and healthy controls. At the examination, the three postfinasteride patients reported muscular stiffness, cramps, tremors, and chronic fatigue in the absence of clinical evidence of any muscular disorder or strength reduction. Severity and frequency of the anxious/depressive symptoms were quite variable; overall, all the subjects had a fairly complex and constant neuropsychiatric pattern. Assessment of neuroactive steroid levels in patients showed some interindividual differences. However, the most important finding was the comparison of their neuroactive steroid levels with those of healthy controls. Indeed, decreased levels of tetrahydroprogesterone, isopregnanolone and dihydrotestosterone and increased levels of testosterone and 17β-estradiol were reported in cerebrospinal fluid of postfinasteride patients. Moreover, decreased levels of dihydroprogesterone and increased levels of 5α-androstane-3α,17β-diol and 17β-estradiol were observed in plasma. The present observations confirm that an impairment of neuroactive steroid levels, associated with depression symptoms, is still present in androgenic alopecia patients treated with finasteride despite the discontinuation of the treatment.

Simultaneous determination of multiple amino acids in plasma in critical illness by high performance liquid chromatography with ultraviolet and fluorescence detection

There is increasing recognition that the host response to critical illness includes derangement of multiple amino acid pathways, including amino acids (AAs) central to metabolism and immune, endothelial and neurological function. To characterise concentration changes of these plasma amino acid we report the development and validation of a method for the quantification of AAs in small volumes of plasma (50μL) using HPLC with simultaneous UV and fluorescence (FL) detection. Protein precipitation and pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) is followed by reversed phase HPLC separation. Calibration curves were built with norleucine as an internal standard. Thirty-three (including the 20 proteinogenic) AAs, were selected as standards and their corresponding concentrations in the plasma of healthy human controls and patients with severe falciparum malaria were quantified. This method enables the detection of perturbations in arginine metabolism, aromatic amino acid pathways, methionine transsulfuration and transmethylation pathways and other metabolic pathways.

SP0025 Neutrophil microparticles - what do they do?

Microparticles (MPs) are small vesicles shed from the plasma membrane that retain unique markers and mediators of their parent cell, enabling them to exert independent functions. Elevated levels of circulating...

Changes of Plasma microRNAs in Heart Transplantation Patients Do Not Reflect microRNA Changes in the Cardiac Allograft Vasculopathy Vessel Wall

Purpose A limiting factor for long term survival after heart transplantation (HTx) is cardiac allograft vasculopathy (CAV). CAV is characterized by hyperplasia of the intima in coronary arteries. Recently the importance of microRNAs (miRs) was described in multiple vascular diseases. The aim of our study was to determine whether changes in miR expression in plasma post HTx reflect changes in the coronary vessel wall of patients with CAV. Methods and Materials Pooled plasma of HTx patients (two pools, each n=3) was analyzed at 6 and 52 weeks after HTx and compared to healthy control plasma. CAV vessels (n=6) were obtained after autopsy and the intimal layer was isolated by tissue laser microdissection. MiR expression in plasma and vessel wall was determined using Q-PCR arrays. Eight miRs were selected from both array analyses (plasma & vessel wall). Criteria for selection were: fold change >2x, Cq-values Results Array analyses showed miR expression changes over time after HTx and in intimal tissue. Within the Q-PCR validation of plasma 1 miR (1/8) showed a significant up-regulation over time (miR-132). The intimal tissue analysis showed 5 miRs (5/8) to have a significant up- (miR-21, -223, and -146b-5p) or down-regulation (miR-886-5p and -214). Within the top 8 of plasma and intima array analysis no overlap was found in the detected miRs. Conclusions MiRs in CAV patients do alter both in plasma and vessel wall. However, the changes in plasma do not reflect changes in the CAV vessel wall. Q-PCR validation on tissue samples confirmed more miRs than validation in plasma, indicating that miRs in tissue show a more representative pattern in array analysis. The miRs selected from the neo-intimal layer might have a role in CAV development and could be considered potential therapeutic targets in the future.

Autoantibodies against Muscarinic Type 3 Receptor in Sjögren's Syndrome Inhibit Aquaporin 5 Trafficking

Sjögren's syndrome (SjS) is a chronic autoimmune disease that mainly targets the salivary and lacrimal glands. It has been controversial whether anti-muscarinic type 3 receptor (α-M3R) autoantibodies in patients with SjS inhibit intracellular trafficking of aquaporin-5 (AQP5), water transport protein, leading to secretory dysfunction. To address this issue, GFP-tagged human AQP5 was overexpressed in human salivary gland cells (HSG-hAQP5) and monitored AQP5 trafficking to the plasma membrane following carbachol (CCh, M3R agonist) stimulation. AQP5 trafficking was indeed mediated by M3R stimulation, shown in partial blockage of trafficking by M3R-antagonist 4-DAMP. HSG-hAQP5 pre-incubated with SjS plasma for 24 hours significantly reduced AQP5 trafficking with CCh, compared with HSG-hAQP5 pre-incubated with healthy control (HC) plasma. This inhibition was confirmed by monoclonal α-M3R antibody and pre-absorbed plasma. Interestingly, HSG-hAQP5 pre-incubated with SjS plasma showed no change in cell volume, compared to the cells incubated with HC plasma showing shrinkage by twenty percent after CCh-stimulation. Our findings clearly indicate that binding of anti-M3R autoantibodies to the receptor, which was verified by immunoprecipitation, suppresses AQP5 trafficking to the membrane and contribute to impaired fluid secretion in SjS. Our current study urges further investigations of clinical associations between SjS symptoms, such as degree of secretory dysfunction, cognitive impairment, and/or bladder irritation, and different profiles (titers, isotypes, and/or specificity) of anti-M3R autoantibodies in individuals with SjS.

Erythrocyte Adhesion in Hemodialysis Patients – A Novel Potential Contributor to Inflammation in End-Stage Renal Disease.

AbstractAbstract 2092 Background:Widespread adhesion of erythrocytes in the vasculature would be incompatible with life. However, in illnesses such as sickle cell disease, malaria and diabetes, adhesive RBCs have been documented and are likely contributors to disease severity, inflammation and coagulation activation. End-stage renal disease is characterized by profound, global inflammation and relative thrombophilia. While we, and others, have reported the extensive exposure of RBC phosphatidylserine in patients on hemodialysis, the role of RBC adhesion in inflammation has never been examined in this condition. We performed an analysis of RBCs from hemodialysis patients to characterize their ability to adhere to other cells, elucidate the potential mechanisms of adhesion, and to relate RBC adhesion to the inflammatory state of hemodialysis. Methods:This study was conducted in a cohort of 20 African American patients receiving in-center hemodialysis at 2 separate dialysis clinics as part of a study of sickle cell trait in hemodialysis. There were 9 of 20 patients with documented sickle cell trait. Blood samples were drawn immediately pre-dialysis and prior to administration of heparin. Plasma samples were processed to minimize platelet activation, aliquoted, and frozen for batch analysis. Plasma factors (RANTES, CRP & CD40L) were analyzed via ELISA. Adhesion events in whole blood were detected via two color flow cytometry. Cause of renal failure, mode of venous access and relevant clinical data were obtained from patient charts. All adhesion assays with washed blood cells were conducted under static conditions using microvascular endothelial cells. All studies were compared to those results of healthy control patients (n =11). Spearman’s regression analysis was performed to analyze the correlation between continuous variables. Mann-Whitney U and Kruskal Wallis tests were used to compare continuous variables between groups. A p<0.05 was considered significant. Results:We found that RBCs from patients on hemodialysis were significantly more adhesive than those from healthy controls. In whole blood, we detected marked RBC adhesion to T-cells (median % of T-cells bound to RBCs: 67.9 [hemodialysis] vs. 10.55 [healthy control]) and platelets (median % of platelets bound to RBCs: 33.0 vs. 1.1%). We also noted significant RBC adhesion to neutrophils (median % of neutrophils bound to RBCs: 11.0% vs. 0.1%). Incubation of healthy RBCs with plasma from hemodialysis patients, but not healthy control plasma, was sufficient to induce RBC adhesion to cultured endothelial cells (median RBCs/mm2: 8.0 vs. 0.5) and to T-cells (median % T-cells bound to RBCs: 30.0 vs. 5.0). Plasma from hemodialysis patients also induced phosphatidylserine exposure on healthy RBCs. Phosphatidylserine exposure on the RBC appeared to mediate RBC adhesion to endothelial cells as annexin V significantly reduced RBC adherence (8.0 vs 3.8 RBC/mm2). The extent to which RBCs in hemodialysis patients adhered to T-cells directly correlated with both plasma RANTES (rspearman = 0.65, 95% CI: 0.247–0.864) and CD40L levels (rspearman= 0.60, 95% CI: 0.1645 – 0.847), but not plasma CRP levels. There was also no significant difference in adhesion of RBCs due to cause of renal failure (diabetes, hypertension, or glomerulonephritis) or presence of sickle cell trait. Conclusions:We describe for the first time a novel adhesion of RBCs in patients receiving hemodialysis and how this adhesion may relate to inflammation in these patients. Our findings also suggest that factors in uremic plasma are sufficient to induce phosphatidylserine exposure on RBCs. This exposure, in addition to providing a site for thrombin generation, also serves as an adhesive moiety on RBCs for endothelial cells. These data may describe an unrecognized etiology of inflammation in hemodialysis and end-stage renal disease. Disclosures:No relevant conflicts of interest to declare.

Comparison between alpha-galactosidase A activity in blood samples collected on filter paper, leukocytes and plasma

ObjectivesTo compare alpha-galactosidase A activity in dried blood spots on filter paper, plasma, and leukocytes of Fabry disease patients and healthy controls, and to develop a miniaturization approach of the techniques to measure activity using plasma and leukocytes. Design and MethodsBlood was collected from healthy controls and Fabry disease patients. Two drops were spotted on filter paper. Plasma and leukocytes were separated from the remaining sample. Enzyme activity was assessed by fluorometry. ResultsSignificant positive correlation between standard and miniaturized techniques was observed. Alpha-galactosidase activity differed for male and female subjects when analyzed using filter paper and plasma. New reference and cutoff values were established based on the differences in alpha-galactosidase activity between genders. A good correlation was observed across biological materials assessed. ConclusionsThe establishment of specific values for men and women increases reliability of commonly used techniques to screen and diagnose Fabry disease.

Relationship between Inflammatory Mediators, A? Levels and ApoE Genotype in Alzheimer Disease

Activation of inflammatory processes is observed within the brain as well as periphery of subjects with Alzheimer's disease (AD). Whether or not inflammation represents a possible cause of AD or occurs as a consequence of the disease process, or, alternatively, whether the inflammatory response might be beneficial to slow the disease progression remains to be elucidated. The cytokine IL-18 shares with IL-1 the same pro-inflammatory features. Consequent to these similarities, IL-18 and its endogenous inhibitor, IL-18BP, were investigated in the plasma of AD patients versus healthy controls (HC). An imbalance of IL-18 and IL-18BP was observed in AD, with an elevated IL-18/IL-18BP ratio that might be involved in disease pathogenesis. As part of the inflammatory response, altered levels of RANTES, MCP-1 and ICAM- 1, molecules involved in cell recruitment to inflammatory sites, were observed in AD. Hence, correlations between IL-18 and other inflammatory plasma markers were analyzed. A negative correlation was observed between IL-18 and IL-18BP in both AD and HC groups. A positive correlation was observed between IL-18 and ICAM-1 in AD patients, whereas a negative correlation was evident in the HC group. IL-18 positively correlated with Aβ in both groups, and no significant correlations were observed between IL-18, RANTES and MCP-1. An important piece of evidence supporting a pathophysiologic role for inflammation in AD is the number of inflammatory mediators that have been found to be differentially regulated in AD patients, and specific ones may provide utility as part of a biomarker panel to not only aid early AD diagnosis, but follow its progression.

Abstract 695: Significant loss of nuclear expression of Actinin-4 in metastatic breast carcinoma and lymph nodes: A novel biomarker for metastatic breast carcinoma

Abstract Background: Actinin-4 is an actin cross-linked protein. The expression and subcellular distribution of actinin-4 (ACTN4) has been associated with multiple roles in tumorigenicity and cancer metastasis. In the present study, we developed a novel antibody against human actinin-4 and investigated the clinical utility of actinin-4 as a biomarker in metastatic breast cancer. Methods and findings: We generated a novel monoclonal antibody (AB-Actinin-4) using hybridoma technology and native ACTN4 protein as an immunogen. The antibody specifically recognized both native and denatured human Actinin-4 protein and displayed high affinity as assessed by ELISA, immunohistochemistry assay, western blot and flow cytometry. Using this antibody and ELISA as well as immunohistochemical techniques, we examined plasma levels and tissue expression of actinin-4 in healthy controls’ and breast cancer patients’ samples. 50 plasma samples from healthy controls and breast cancer patients were analyzed for the presence of soluble actinin-4. ELISA results showed significantly decreased actinin-4 levels in plasma samples from patients with breast carcinomas compared to healthy control plasma. A total of 156 cases, including 70 normal, 36 non-metastatic breast carcinoma, 50 breast carcinoma tissues with regional metastasis, and 50 matched metastatic lymph nodes were examined. The immunoreactivity of actinin-4 was located mainly in nucleus and cytoplasm of cells. To assess the difference between groups, the staining intensity and intracellular distribution were compared and the intensity in nucleus and cytoplasm was separately scored as 0, 1+, 2+ and 3+. Grades 0 and 1+ were defined as negative and 2+ and 3+ as positive. Significant differences between the groups were found in nuclear staining. 92% of normal and 89% of non-metastatic breast carcinoma samples displayed positive nuclear staining. In contrast, only 29% of metastatic lymph nodes and 51% of metastatic breast cancer tissues showed strong nuclear staining. Conclusion: Actinin-4 demonstrated significantly decreased expression in plasma from breast cancer patients. Metastasis leads to significant loss of the nuclear expression of Actinin-4 in breast cancer tissues, in comparison to normal and non-metastatic breast carcinoma tissues. Our data suggests that Actinin-4 might have significant impact in breast cancer metastasis. Actinin-4 may serve as a reliable biomarker for the diagnosis and prognosis of metastatic breast cancer as well as could be a therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 695. doi:1538-7445.AM2012-695

Detection of allantoin in clinical samples using hydrophilic liquid chromatography with stable isotope dilution negative ion tandem mass spectrometry

Allantoin is the major oxidation product of urate in humans and is a potential biomarker of oxidative stress. Several methods are used to measure allantoin in biological samples but they have inherent issues that can include lack of specificity and sensitivity, difficulty in sample preparation, or artefactual generation of allantoin. We have developed a method for measuring allantoin using hydrophilic liquid chromatography with stable isotope dilution tandem mass spectrometry (HILIC–MS/MS). It was validated for measuring allantoin in plasma, synovial fluid and urine from human subjects. The limit of quantification was determined to be 10fmol and the assay displayed excellent linearity for the wide range of concentrations found in clinical samples. Relative standard deviations were <5% for between-day and <7% for within-day variation. Accuracy was between 100% and 104%. Concentrations of allantoin in plasma of healthy controls (2.0μM; interquartile range 1.4–3.6μM, n=35) was significantly lower (p<0.001) than that in plasma from patients with rheumatoid arthritis (3.7μM; IQR 3.0–5.6μM, n=43) and in synovial fluid of patients with gout (3.3μM; IQR 2.8–5.8μM, n=10). This newer HILIC–MS/MS method is a simple and highly sensitive assay for detection of allantoin. It can be used to assess the level of oxidative stress in human pathologies.

Plasma β-Amyloid Levels in Cerebral Amyloid Angiopathy-Associated Hemorrhagic Stroke

Background: Cerebral amyloid angiopathy (CAA) is one of the main causes of intracerebral hemorrhage (ICH) in the elderly. Objective: To analyze β-amyloid (Aβ) species in plasma in order to uncover biological markers that may contribute to improve the diagnosis of CAA in life. Methods: We determined the level of Aβ(1–40), Aβ(N–40), Aβ(1–42) and Aβ(N–42) in plasma of CAA-related ICH patients (n = 29) and healthy controls (n = 21) using xMAP® technology. Hemorrhages were identified and classified using a CT scan and brain MRI. Patients were clinically classified as probable or possible CAA according to the Boston criteria. Results: We found that plasma full-length Aβ(1–42) and truncated fragments Aβ(N–42) were higher in probable CAA patients than in controls (p < 0.001 and p = 0.046, respectively), and full-length Aβ(1–40) was selectively elevated in probable CAA compared to possible cases (p = 0.015) and controls (p = 0.005). In addition, plasma Aβ(N–42) levels were also higher in patients that presented multiple lobar macrohemorrhages compared to patients that had one symptomatic hemorrhagic event (p = 0.022), indicating that a certain degree of CAA severity is necessary to show increased Aβ fragments in peripheral circulation. Conclusion: Our results suggest that specific Aβ fragments in plasma might be considered as potential biomarkers for the diagnosis of CAA.

A novel neutrophil derived inflammatory biomarker of pulmonary exacerbation in cystic fibrosis

BackgroundThe focus of this study was to characterize a novel biomarker for cystic fibrosis (CF) that could reflect exacerbations of the disease and could be useful for therapeutic stratification of patients, or for testing of potential drug treatments. This study focused exclusively on a protein complex containing alpha-1 antitrypsin and CD16b (AAT:CD16b) which is released into the bloodstream from membranes of pro-inflammatory primed neutrophils. MethodsNeutrophil membrane expression and extracellular levels of AAT and CD16b were quantified by flow cytometry, Western blot analysis and by 2D-PAGE. Interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha) and AAT:CD16b complex were quantified in CF plasma (n=38), samples post antibiotic treatment for 14days (n=10), chronic obstructive pulmonary disease (n=10), AAT deficient (n=10) and healthy control (n=14) plasma samples by ELISA. ResultsCell priming with IL-8 and TNF-alpha caused release of the AAT:CD16b complex from the neutrophil cell membrane. Circulating plasma levels of IL-8, TNF-alpha and AAT:CD16b complex were significantly higher in patients with CF than in the other patient groups or healthy controls (P<0.05). Antibiotic treatment of pulmonary exacerbation in patients with CF led to decreased plasma protein concentrations of AAT:CD16b complex with a significant correlation with improved FEV1 (r=0.81, P=0.003). ConclusionThe results of this study have shown that levels of AAT:CD16b complex present in plasma correlate to the inflammatory status of patients. The AAT:CD16b biomarker may become a useful addition to the clinical diagnosis of exacerbations in CF.

Investigation of Matrix Metalloproteinases, MMP-2 and MMP-9, in Plasma Reveals a Decrease of MMP-2 in Alzheimer's Disease

Pathological changes in the Alzheimer's disease (AD) brain include amyoid-β (Aβ) plaques, and neurofibrillary tangles, as well as neuronal death and synaptic loss. Matrix metalloproteinases MMP-2 and MMP-9 are known to degrade Aβ, and their expressions are increased in the AD brain, in particular in the astrocytes surrounding amyloid plaque. To investigate a possible association between plasma metalloproteinases and AD, we quantified MMP-2 and MMP-9 activities in the plasma of healthy controls (HC, n = 56), cases with mild cognitive impairment (MCI, n = 45), and AD (n = 50). All cases had previously been imaged with Pittsburgh compound B (PiB) and had a Mini-Mental Status Examination (MMSE) assessment. MMP-2 and MMP-9 activity was determined using gelatine-zymography. There was a significant 1.5-fold decrease in MMP-2 activity in the AD group compared to HC (p < 0.001) and a 1.4-fold decrease compared to MCI (p < 0.01). There was no difference in MMP-9 levels between the three groups. A positive correlation was identified between MMP-2 plasma activity and MMSE score (r = 0.16, p < 0.05), but there was no association with PiB. This is the first report of a change in MMP-2 activity in AD plasma and these findings may provide some insight into AD pathogenesis.

Decreased oxidative phosphorylation and PGAM deficiency in horses suffering from atypical myopathy associated with acquired MADD

Earlier research on ten horses suffering from the frequently fatal disorder atypical myopathy showed that MADD (multiple acyl-CoA dehydrogenase deficiency) is the biochemical derangement behind atypical myopathy. From five horses that died as a result of this disease and seven healthy control horses, urine and plasma were collected ante mortem and muscle biopsies were obtained immediately post-mortem (2 patients and 7 control horses), to analyse creatine, purine and carbohydrate metabolism as well as oxidative phosphorylation.In patients, the mean creatine concentration in urine was increased 17-fold and the concentration of uric acid approximately 4-fold, compared to controls.The highest degree of depletion of glycogen was observed in the patient with the most severe myopathy clinically. In this patient, glycolysis was more active than in the other patients and controls, which may explain this depletion. One patient demonstrated very low phosphoglycerate mutase (PGAM) activity, less than 10% of reference values.Most respiratory chain complex activity in patients was 20–30% lower than in control horses, complex II activity was 42% lower than normal, and one patient had severely decrease ATP-synthase activity, more than 60% lower than in control horses.General markers for myopathic damage are creatine kinase (CK) and lactic acid in plasma, and creatine and uric acid in urine. To obtain more information about the cause of the myopathy analysis of carbohydrate, lipid and protein metabolism as well as oxidative phosphorylation is advised.This study expands the diagnostic possibilities of equine myopathies.

Use of transcriptional signatures induced in lymphoid and myeloid cell lines as an inflammatory biomarker in Type 1 diabetes

Inflammation is common to many disorders and responsible for tissue and organ damage. In many disorders, the associated peripheral cytokine milieu is dilute and difficult to measure, necessitating development of more sensitive and informative biomarkers for mechanistic studies, earlier diagnosis, and monitoring therapeutic interventions. Previously, we have shown that plasma of recent-onset (RO) Type 1 diabetes patients induces a disease-specific proinflammatory transcriptional profile in fresh peripheral blood mononuclear cells (PBMC) compared with that of healthy controls (HC). To eliminate assay variance introduced through the use of multiple donors or multiple draws of the same person over time, we evaluated human leukemia cell lines as potential surrogates for fresh PBMC. We 1) tested seven different cell lines in their power to differentiate RO from HC plasma and 2) compared the similarity of the signatures generated across the seven cell lines to that obtained with fresh PBMC. While each cell line tested exhibited a distinct transcriptional response when cultured with RO or HC plasma, the expression profile induced in any single cell line shared little identity with that of the other cell lines or fresh PBMC. In terms of regulated biological pathways, the transcriptional response of each cell line shared varying degrees of functional identity with fresh PBMC. These results indicate that use of human leukemia cell lines as surrogates for fresh PBMC has potential in detecting perturbations to the peripheral cytokine milieu. However, the response of each is distinct, possessing varying degrees of functional relatedness to that observed with PBMC.