Capillary blood self-collection for high-throughput proteomics.

In this study, we sought to compare protein concentrations obtained from a high-throughput proteomics platform (Olink) on samples collected using capillary blood self-collection (with the Tasso+ device) versus standard venipuncture (control). Blood collection was performed on 20 volunteers, including one sample obtained via venipuncture and two via capillary blood using the Tasso+ device. Tasso+ samples were stored at 2°C-8°C for 24-hs (Tasso-24) or 48-h (Tasso-48) prior to processing to simulate shipping times from a study participant's home. Proteomics were analyzed using Olink (384 Inflammatory Panel). Tasso+ blood collection was successful in 37/40 attempts. Of 230 proteins included in our analysis, Pearson correlations (r) and mean coefficient of variation (CV) between Tasso-24 or Tasso-48 versus venipuncture were variable. In the Tasso-24 analysis, 34 proteins (14.8%) had both a correlation r > 0.5 and CV < 0.20. In the Tasso-48 analysis, 68 proteins (29.6%) had a correlation r > 0.5 and CV < 0.20. Combining the Tasso-24 and Tasso-48 analyses, 26 (11.3%) proteins met these thresholds. We concluded that protein concentrations from Tasso+ samples processed 24-48 h after collection demonstrated wide technical variability and variable correlation with a venipuncture gold-standard. Use of home capillary blood self-collection for large-scale proteomics should be limited to select proteins with good agreement with venipuncture.

Protein Identification for Stroke Progression via Mendelian Randomization in Million Veteran Program and UK Biobank.

BACKGROUND: Individuals who have experienced a stroke, or transient ischemic attack, face a heightened risk of future cardiovascular events. Identification of genetic and molecular risk factors for subsequent cardiovascular outcomes may identify effective therapeutic targets to improve prognosis after an incident stroke. METHODS: We performed genome-wide association studies for subsequent major adverse cardiovascular events (MACE; ncases=51 929; ncontrols=39 980) and subsequent arterial ischemic stroke (AIS; ncases=45 120; ncontrols=46 789) after the first incident stroke within the Million Veteran Program and UK Biobank. We then used genetic variants associated with proteins (protein quantitative trait loci) to determine the effect of 1463 plasma protein abundances on subsequent MACE using Mendelian randomization. RESULTS: Two variants were significantly associated with subsequent cardiovascular events: rs76472767 near gene RNF220 (odds ratio, 0.75 [95% CI, 0.64-0.85]; P=3.69×10-8) with subsequent AIS and rs13294166 near gene LINC01492 (odds ratio, 1.52 [95% CI, 1.37-1.67]; P=3.77×10-8) with subsequent MACE. Using Mendelian randomization, we identified 2 proteins with an effect on subsequent MACE after a stroke: CCL27 ([C-C motif chemokine 27], effect odds ratio, 0.77 [95% CI, 0.66-0.88]; adjusted P=0.05) and TNFRSF14 ([tumor necrosis factor receptor superfamily member 14], effect odds ratio, 1.42 [95% CI, 1.24-1.60]; adjusted P=0.006). These proteins are not associated with incident AIS and are implicated to have a role in inflammation. CONCLUSIONS: We found evidence that 2 proteins with little effect on incident stroke appear to influence subsequent MACE after incident AIS. These associations suggest that inflammation is a contributing factor to subsequent MACE outcomes after incident AIS and highlights potential novel targets.

Multiplexed MRM-based proteomics for identification of circulating proteins as biomarkers of cardiovascular damage progression associated with diabetes mellitus.

BACKGROUND: Type 2 diabetes mellitus (T2DM) increases the risk of coronary heart disease (CHD) by 2-4 fold, and is associated with endothelial dysfunction, dyslipidaemia, insulin resistance, and chronic hyperglycaemia. The aim of this investigation was to assess, by a multimarker mass spectrometry approach, the predictive role of circulating proteins as biomarkers of cardiovascular damage progression associated with diabetes mellitus. METHODS: The study considered 34 patients with both T2DM and CHD, 31 patients with T2DM and without CHD, and 30 patients without diabetes with a diagnosis of CHD. Plasma samples of subjects were analysed through a multiplexed targeted liquid chromatography mass spectrometry (LC-MS)-based assay, namely Multiple Reaction Monitoring (MRM), allowing the simultaneous detection of peptides derived from a protein of interest. Gene Ontology (GO) Analysis was employed to identify enriched GO terms in the biological process, molecular function, or cellular component categories. Non-parametric multivariate methods were used to classify samples from patients and evaluate the relevance of the analysed proteins' panel. RESULTS: A total of 81 proteins were successfully quantified in the human plasma samples. Gene Ontology analysis assessed terms related to blood microparticles, extracellular exosomes and collagen-containing extracellular matrix. Preliminary evaluation using analysis of variance (ANOVA) of the differences in the proteomic profile among patient groups identified 13 out of the 81 proteins as significantly different. Multivariate analysis, including cluster analysis and principal component analysis, identified relevant grouping of the 13 proteins. The first main cluster comprises apolipoprotein C-III, apolipoprotein C-II, apolipoprotein A-IV, retinol-binding protein 4, lysozyme C and cystatin-C; the second one includes, albeit with sub-grouping, alpha 2 macroglobulin, afamin, kininogen 1, vitronectin, vitamin K-dependent protein S, complement factor B and mannan-binding lectin serine protease 2. Receiver operating characteristic (ROC) curves obtained with the 13 selected proteins using a nominal logistic regression indicated a significant overall distinction (p < 0.001) among the three groups of subjects, with area under the ROC curve (AUC) ranging 0.91-0.97, and sensitivity and specificity ranging from 85 to 100%. CONCLUSIONS: Targeted mass spectrometry approach indicated 13 multiple circulating proteins as possible biomarkers of cardiovascular damage progression associated with T2DM, with excellent classification results in terms of sensitivity and specificity.

Preclinical development and characterization of a human plasma-derived high-purity factor X concentrate for therapeutic use.

INTRODUCTION: Hereditary factor X deficiency is a rare bleeding disorder, with limited treatment options. This paper describes the approach to pre-clinical development and characterization of a high-purity plasma-derived factor X concentrate, to achieve orphan drug marketing authorization for the treatment of hereditary factor X deficiency. METHODS: A chromatographic process was developed, to purify factor X from human plasma for fractionation. The product was characterized using in vitro, in vivo and ex vivo tests for potency, purity, thrombogenicity, immunogenicity, toxicity and stability. RESULTS: The production process complied with good pharmaceutical manufacturing practice. It achieved 6000-fold purification and 100-fold concentration of the factor X protein compared to human plasma. The factor X protein was 94%-96% pure. Other residual plasma proteins were well below levels in plasma, minimizing potential interference in hemostasis after therapeutic administration. Effective virus-reduction during manufacture, and the absence of thrombogenicity, toxicity and immunogenic potential were confirmed, addressing the main safety concerns historically associated with plasma-derived therapeutics. The freeze-dried product remained stable between +2°C and +30°C for at least three years. After reconstitution with water for injections, the factor X activity was maintained for at least 48 h at +18°C to +22°C. CONCLUSION: Targeted pre-clinical development of the first highly-purified concentrate to treat hereditary factor X deficiency is described. Following international guidelines for nonclinical safety testing, particular strategies were adopted for unmodified products derived from human blood plasma. This approach may also be relevant to the development of other ultra-orphan medicinal products.

Serum Protein Exposure Activates a Core Regulatory Program Driving Human Proximal Tubule Injury.

BACKGROUND: The kidneys efficiently filter waste products while retaining serum proteins in the circulation. However, numerous diseases compromise this barrier function, resulting in spillage of serum proteins into the urine (proteinuria). Some studies of glomerular filtration suggest that tubules may be physiologically exposed to nephrotic-range protein levels. Therefore, whether serum components can directly injure the downstream tubular portions of the kidney, which in turn can lead to inflammation and fibrosis, remains controversial. METHODS: We tested the effects of serum protein exposure in human kidney tubule microphysiologic systems and with orthogonal epigenomic approaches since animal models cannot directly assess the effect of serum components on tubules. RESULTS: Serum, but not its major protein component albumin, induced tubular injury and secretion of proinflammatory cytokines. Epigenomic comparison of serum-injured tubules and intact kidney tissue revealed canonical stress-inducible regulation of injury-induced genes. Concordant transcriptional changes in microdissected tubulointerstitium were also observed in an independent cohort of patients with proteinuric kidney disease. CONCLUSIONS: Our results demonstrate a causal role for serum proteins in tubular injury and identify regulatory mechanisms and novel pathways for intervention.

Diagnostic performance improvement with combined use of proteomics biomarker assay and breast ultrasound.

PURPOSE: To investigate the combined use of blood-based 3-protein signature and breast ultrasound (US) for validating US-detected lesions. METHODS: From July 2011 to April 2020, women who underwent whole-breast US within at least 6 months from sampling period were retrospectively included. Blood-based 3-protein signature (Mastocheck®) value and US findings were evaluated. Following outcome measures were compared between US alone and the combination of Mastocheck® value with US: sensitivity, specificity, positive predictive value (PPV), negative predictive value, area under the receiver operating characteristic curve (AUC), and biopsy rate. RESULTS: Among the 237 women included, 59 (24.9%) were healthy individuals and 178 (75.1%) cancer patients. Mean size of cancers was 1.2 ± 0.8 cm. Median value of Mastocheck® was significantly different between nonmalignant (- 0.24, interquartile range [IQR] - 0.48, - 0.03) and malignant lesions (0.55, IQR - 0.03, 1.42) (P < .001). Utilizing Mastocheck® value with US increased the AUC from 0.67 (95% confidence interval [CI] 0.61, 0.73) to 0.81 (95% CI 0.75, 0.88; P < .001), and specificity from 35.6 (95% CI 23.4, 47.8) to 64.4% (95% CI 52.2, 76.6; P < .001) without loss in sensitivity. PPV was increased from 82.2 (95% CI 77.1, 87.3) to 89.3% (95% CI 85.0, 93.6; P < .001), and biopsy rate was significantly decreased from 79.3 (188/237) to 72.1% (171/237) (P < .001). Consistent improvements in specificity, PPV, and AUC were observed in asymptomatic women, in women with dense breast, and in those with normal/benign mammographic findings. CONCLUSION: Mastocheck® is an effective tool that can be used with US to improve diagnostic specificity and reduce false-positive findings and unnecessary biopsies.

Haptoglobin-related protein in human plasma correlates to haptoglobin concentrations and phenotypes.

Haptoglobin-related protein (Hpr) is a plasma protein with high sequence similarity to haptoglobin (Hp). Like Hp, Hpr also binds hemoglobin (Hb) with high affinity, but it does not bind to the Hb-Hp receptor CD163 on macrophages. The Hpr concentration is markedly lower than Hp in plasma and its regulation is not understood. In the present study, we have developed non-crossreactive antibodies to Hpr to analyze the Hpr concentration in 112 plasma samples from anonymized individuals and compared it to Hp. The results show that plasma Hpr correlated with Hp concentrations (rho = 0.46, p = .0001). Hpr accounts for on average 0.35% of the Hp/Hpr pool but up to 29% at low Hp levels. Furthermore, the Hpr concentrations were significantly lower in individuals with the Hp2-2 phenotype compared to those with the Hp2-1 or Hp1-1 phenotypes. Experimental binding analysis did not provide evidence that Hpr associates with Hp and in this way is removed via CD163. In conclusion, the Hpr concentration correlates to Hp concentrations and Hp-phenotypes by yet unknown mechanisms independent of CD163-mediated removal of Hb-Hp complexes.

Re-evalution of the measurement of haptoglobin in human plasma samples.

Haptoglobin (Hp) is an abundant plasma protein scavenging hemoglobin (Hb) via CD163 on macrophages. This process consumes Hp, which therefore negatively correlates to hemolysis. However, exact measurements of Hp plasma levels are complicated by different phenotypes (Hp1-1, Hp2-1, and Hp2-2) forming different oligomeric states with differences in immunoreactivity. In addition, humans have an immune-cross-reactive Hp-related protein. In the present study, we developed Hp-specific monoclonal antibodies for an accurate Hp analysis of the different Hp phenotypes in a panel of 112 anonymous samples from hospitalized individuals subjected to routine Hp immunoturbidimetric measurements. The data revealed immunoturbidimetry as a reliable method in most cases but also that the use of non-phenotype-specific calibrators leads to substantial bias in the measurement of the Hp-concentration, non at least in Hp1-1 individuals. Furthermore, analysis of the Hb-dependence of the CD163 interaction with Hp1-1 and Hp2-2 showed that a higher 'cost-effectiveness' in the consumption of dimeric Hp1-1 versus multimeric Hp phenotypes is a likely contribution to the observed differences in the plasma levels of the Hp phenotypes. In conclusion, the determination of Hp phenotype and the use of phenotype-specific calibrators are essential to obtain a precise estimate of the Hp level in healthy and diseased individuals.

Metabolic syndrome and the plasma proteome: from association to causation.

BACKGROUND: The metabolic syndrome (MetS), defined by the simultaneous clustering of cardio-metabolic risk factors, is a significant worldwide public health burden with an estimated 25% prevalence worldwide. The pathogenesis of MetS is not entirely clear and the use of molecular level data could help uncover common pathogenic pathways behind the observed clustering. METHODS: Using a highly multiplexed aptamer-based affinity proteomics platform, we examined associations between plasma proteins and prevalent and incident MetS in the KORA cohort (n = 998) and replicated our results for prevalent MetS in the HUNT3 study (n = 923). We applied logistic regression models adjusted for age, sex, smoking status, and physical activity. We used the bootstrap ranking algorithm of least absolute shrinkage and selection operator (LASSO) to select a predictive model from the incident MetS associated proteins and used area under the curve (AUC) to assess its performance. Finally, we investigated the causal effect of the replicated proteins on MetS using two-sample Mendelian randomization. RESULTS: Prevalent MetS was associated with 116 proteins, of which 53 replicated in HUNT. These included previously reported proteins like leptin, and new proteins like NTR domain-containing protein 2 and endoplasmic reticulum protein 29. Incident MetS was associated with 14 proteins in KORA, of which 13 overlap the prevalent MetS associated proteins with soluble advanced glycosylation end product-specific receptor (sRAGE) being unique to incident MetS. The LASSO selected an eight-protein predictive model with an (AUC = 0.75; 95% CI = 0.71-0.79) in KORA. Mendelian randomization suggested causal effects of three proteins on MetS, namely apolipoprotein E2 (APOE2) (Wald-Ratio = - 0.12, Wald-p = 3.63e-13), apolipoprotein B (APOB) (Wald-Ratio = - 0.09, Wald-p = 2.54e-04) and proto-oncogene tyrosine-protein kinase receptor (RET) (Wald-Ratio = 0.10, Wald-p = 5.40e-04). CONCLUSIONS: Our findings offer new insights into the plasma proteome underlying MetS and identify new protein associations. We reveal possible casual effects of APOE2, APOB and RET on MetS. Our results highlight protein candidates that could potentially serve as targets for prevention and therapy.

Protein Electrophoresis of Serum and Heparinized Plasma in the Common Mynah (Acridotheres tristis).

Although serum protein electrophoresis is a diagnostic tool available through many veterinary laboratories, there currently are no reference intervals for protein fractions in healthy common mynahs (Acridotheres tristis). Therefore, electrophoretic patterns of proteins in serum and heparinized plasma of the common mynah were evaluated. Blood specimens were collected from 55 healthy adult common mynahs of unknown age (26 males and 29 females). The serum total protein and protein fractions were measured using the biuret method followed by cellulose acetate electrophoresis (CAE). The serum level of albumin was compared with bromocresol green (BCG) dye-binding and CAE methods. Four protein fractions, including albumin and α, ß, and γ globulins, were recorded in the electrophoretogram of serum specimens. Sex appeared to have no significant effect on the measured parameters. The serum BCG albumin fraction was significantly higher than the CAE albumin fraction (P = .01). Also, the comparison of total protein and protein fractions in serum and plasma specimens of 25 of the 55 birds sampled showed that total protein (Cohen index d = 0.66, P = .03), gamma globulin (d = 1.13, P = .00), and total globulin (d = 0.67, P = .00) in plasma samples were significantly higher than those in serum samples. The results of this study provide the specific reference intervals for total protein and protein fractions in common mynahs, which are essential for proper interpretation of laboratory results and also revealed that the albumin measurement by the BCG method yields unreliable results in common mynahs.

[Effect and underling mechanism of 6% hydroxyethyl starch 130/0.4 on serum albumen in trauma orthopedic patients during operation].

Objective: To investigate the effect of 6% hydroxyethyl starch 130/0.4(HES) on protein in severe trauma orthopedic patients after acute hemodilution. Methods: Fourty-eight severe trauma patients who met the inclusion criteria were selected from June 2018 to December 2018 in Yantaishan Hospital, and were randomly divided into two groups (n=24): group A and group B. Group A was ringer's sodium lactate control group, and group B was HES treatment group. After the tracheal intubation, the patients of group A were infused with 10% blood volume of sodium lactate ringer at 0.5 ml·kg(-1)·min(-1), and the patients in group B were infused with 10% blood volume of HES at 0.5 ml·kg(-1)·min(-1). Total protein (TP), human serum albumin (HSA), numbers of circulating endothelium cells (CEC), C-reactive protein (CRP), and serum levels of tumor necrosis factor-alpha (TNF-α), interleukin (IL)-10 and IL-6 were measured immediately after acute hemodilution (T(0)), 24 hours (T(1)) and 48 hours (T(2)) after acute hemodilution. After infusion into human body, HES bond to HSA, and fluorescence spectroscopy was used to analyze the binding relationship between HES and HSA in order to further study the effects of HES on HSA. Results: The HSA, TP, CEC, TNF-α, IL-6, IL-10, CRP at T(0) of group A were (38±5) g/L, (66±5) g/L, (5.5±0.4)/0.9 µl, (24±5) µg/L, (8.9±0.8) µg/L, (44±6) µg/L, (13.6±1.4) mg/L; While at T(1) were (33±5) g/L, (60±6) g/L, (10.2±0.7)/0.9 µl, (87±9) µg/L, (38.8±2.3) µg/L, (57±7) µg/L, (23.4±2.4) mg/L. The HSA, TP, CEC, TNF-α, IL-6, IL-10, CRP at T(0) of group B were(38±4)g/L, (66±5) g/L, (5.4±0.6)/0.9 µl, (24±6) µg/L, (9.1±0.9) µg/L, (45±6) µg/L, (13.4±1.8) mg/L; While at T(1) were (35±5)g/L, (62±5)g/L, (7.4±0.6)/0.9 µl, (70±8) µg/L, (29.5±3.1) µg/L, (72±6) µg/L, (19.7±2.2) mg/L. HSA and TP decreased at T(1) in group A as compared with T(0) (P<0.05), contrarily CEC increased significantly at T(1), TNF-ɑ, IL-6, IL-10 and CRP augmented at T(1) and T(2) in two groups (P<0.05). In comparison with the patients of group A, CEC decreased significantly at T(1) (P<0.05). TNF-ɑ, IL-6, CRP reduced significantly at T(1) and T(2) (P<0.05), but IL-10 increased at T(1) and T(2) in group B (P<0.05). The secondary structure of HSA changed after HES was added in the HES solution. The fluorescence intensity of HSA decreased with the increase of HES concentration,which suggested that HES induced HSA fluorescence quenching. HES could bind to Trp-214 residue in HSA at a molecular ration of 1∶1. Conclusions: 6% HES reduces the occurrence of low protein level in severe trauma patients after operation. HES could bind to Trp-214 amino acid residue in HSA and form the complex at a molecular ratio of 1∶1. The formation of HES-HSA complex increases the volume of HES, avoids the vascular leakage, protects the vascular endothelial cells, and induces anti-inflammatory immunity in the patients with capillary syndrome.

How Full-Length FVIII Benefits from Its Heterogeneity - Insights into the Role of the B-Domain.

PURPOSE: To explore how the natural heterogeneity of human coagulation factor VIII (FVIII) and the processing of its B-domain specifically modulate protein aggregation. METHODS: Recombinant FVIII (rFVIII) molecular species containing 70% or 20% B-domain, and B-domain-deleted rFVIII (BDD-rFVIII), were separated from full-length recombinant FVIII (FL-rFVIII). Purified human plasma-derived FVIII (pdFVIII) was used as a comparator. Heterogeneity and aggregation of the various rFVIII molecular species, FL-rFVIII and pdFVIII were analysed by SDS-PAGE, dynamic light scattering, high-performance size-exclusion chromatography and flow cytometry-based particle analysis. RESULTS: FL-rFVIII and pdFVIII were heterogeneous in nature and demonstrated similar resistance to aggregation under physical stress. Differences were observed between these and among rFVIII molecular species. FVIII molecular species exhibited diverging aggregation pathways dependent on B-domain content. The propensity to form aggregates increased with decreasing proportions of B-domain, whereas the opposite was observed for oligomer formation. Development of cross-ß sheet-containing aggregates in BDD-rFVIII induced effective homologous seeding and faster aggregation. Naturally heterogeneous FL-rFVIII and pdFVIII displayed the lowest propensity to aggregate in all experiments. CONCLUSIONS: These results demonstrate that pdFVIII and FL-rFVIII have similar levels of molecular heterogeneity, and suggest that heterogeneity and the B-domain are involved in stabilising FVIII by modulating its aggregation pathway.

[Efficacy of deproteinized calf blood extract eye drops on early recovery after pterygium surgery].

Objective: To investigate the effect of deproteinized calf blood extract eye drops on early postoperative recovery in primary pterygium patients. Methods: This is a prospective randomized controlled study. Patients diagnosed with primary pterygium in single eye at affiliated Xiamen Eye Center of Xiamen University during March 2016 to May 2016 were enrolled. After Pterygium excision with autologous conjunctival transplantation, patients were randomly assigned into four groups by a random number table, treated with anti-inflammaroty drugs only (control group) or combined with the following agents: deproteinized calf blood extract eye drops (DCBE group), carboxymethylcellulose sodium eye drops (CMC group), and recombinant human epidermal growth factor eye drops (rEGF group). Short-form McGill pain questionnaire, slit lamp and corneal fluorescein sodium staining, non-contact intraocular pressure, uncorrected visual acuity (UCVA) and best corrected visual acquity (BCVA) as well as redness score of bulbar conjunctiva were performed before surgery (d0) and on day 1 (d1), day 2 (d2), day 3 (d3), day 7 (d7) and day 14 (d14) after surgery. Results: One hundred and fourteen patients including 43 males and 71 females, aged (48.9±12.5) years, were eventually included in this study. The McGill scores gradually decreased after surgery in all groups. On d2, the McGill score in DCBE group, control group, CMC group and rEGF group was (1.42±0.67), (2.21±0.88), (1.93±1.08) and (1.77±1.18), respectively; On d3, the score was (1.32±0.54), (1.93±0.72), (1.79±0.87) and (1.52±0.77), respectively. On d2 and d3, statistical difference was recorded among groups (d2, F=3.43, P=0.019; d3, F=4.047, P=0.009), and the McGill score of DCBE group was significantly lower than that of CMC group (d2, P=0.047, d3, P=0.017). On d2, the percentage of corneal epithelium defect in DCBE group, control group, CMC group and rEGF group was 8.6%±1.9%, 11.7%±1.7%, 11.5%±1.9% and 10.4%±1.8%, respectively; On d3, the percentage was 4.5%±2.2%, 9.2%±2.4%, 7.4%±2.5% and 5.9%±2.3%, respectively. On d2 and d3, statistical difference of corneal epithelium defect percentage was recorded among groups (d2, F=17.17, P<0.001; d3, F=21.4, P<0.001). On d2, the percentage of corneal epithelium defect in DCBE group was significantly lower than the other three groups (P<0.01); On d3, the percentage of corneal epithelium defect in DCBE group was significantly lower than control group and CMC group (P<0.001), while no difference was found between DCBE group and rEGF group (P>0.05). However, no statistical differences were recorded in the number of patients with vision improvement among the groups (P>0.05). The intraocular pressure remained stable. No differences in the conjunctival redness score were found among the groups after surgery (P>0.05). Conclusion: Our data demonstrated the efficacy of deproteinized calf blood extract eye drops on the postoperative management in patients with primary patients, with faster pain relief and promoted epithelium recovery. (Chin J Ophthalmol, 2019, 55:134-140).

Calprotectin (S100A8/A9) should preferably be measured in EDTA-plasma; results from a longitudinal study of patients with rheumatoid arthritis.

Calprotectin (S100A8/A9), a protein expressed in neutrophils and monocytes/macrophages in circulation and inflamed tissue, is associated with measures of disease activity in rheumatoid arthritis (RA) patients both when measured in ethylenediaminetetraacetic acid (EDTA)-plasma and in serum. We wanted to explore if EDTA-plasma or serum should be preferred for calprotectin as a marker of disease activity. Calprotectin was analysed in EDTA-plasma and serum by enzyme-linked immunosorbent assay (ELISA) at baseline in 141 RA patients, starting biologic disease-modifying anti-rheumatic drugs (bDMARDs), and after three months. Differences between plasma and serum levels of calprotectin were assessed by Wilcoxon signed rank test. Variability was assessed by quartile coefficient of dispersion. Spearman's test explored correlations between calprotectin in plasma and serum and between calprotectin (plasma or serum) and clinical/ultrasound (US) measures of disease activity. Bland Altman plots were used for method comparisons. Conventional inflammatory markers were evaluated for comparison. Calprotectin had similar variability when measured in plasma and serum, but there was a significant difference in concentrations between plasma and serum (p < .001). The correlation coefficients at baseline between calprotectin measured in plasma/serum and measures of disease activity were rs = 0.62/0.46 for sum power Doppler score (PD), rs = 0.60/0.48 for assessor's global visual analogue scale (VAS), rs = 0.59/0.43 for sum grey scale (GS) score and rs = 0.47/0.37 for swollen joint count of 32, all p < .001. Similar differences were found after three months. Calprotectin measured in plasma showed the strongest associations with assessments of disease activity, and EDTA-plasma should preferably be used when evaluating disease activity in RA patients.

[Anemia in the aged]. / Anämie im Alter.

Anemia in the aged is still often an underestimated clinical problem; however, in recent years increasing research on this topic has permitted deeper insights, allowing a differentiated approach to anemia in the aged. Meanwhile, multicausality and a negative impact on functional outcome have become characteristics of anemia in older people. This has led to a scientific discussion on the question of accepting anemia as a geriatric syndrome. The present article gives a concise overview of the current state of research on this clinically relevant subject.

Milk production and blood metabolites of dairy cattle as influenced by thermal-humidity index.

The effects of high thermal stress on serum protein metabolites, milk production of transition dairy cows in semi-arid areas in South Africa were evaluated. Forty, ± 8 months pregnant, Jersey heifers (± 26 months) in zero grazing management were selected during summer from two semi-arid communal areas. Summer thermal-humidity index (THI) of the areas were THI-1 (72-83: extreme caution) and THI-2 (75-87: danger). Blood samples were collected (21 days pre-partum, and 21 and 75 days post-partum) and analysed for serum protein metabolites. Milk yield was recorded daily and samples collected for milk fat, protein, lactose and urea nitrogen analysis. Heifers in THI-2 had lower (P < 0.05) total serum proteins, albumin and blood urea nitrogen than THI-1. Post-calving, cows in THI-1 had higher (P < 0.05) TP (73.4 vs 67.9 g/l) and BUN (4.61 vs 3.77 mmol/l) at 21 DIM, and lower (P creatinine at 21 and 75 DIM than THI-2 group. Milk yield, fat and protein in THI-2 were all lower (P < 0.05) than THI-1 21DIM. The results confirm that heat stress affects utilisation of nutrients in transition dairy cows.

Is cardiovascular disease in patients with diabetes associated with serum levels of MMP-2, LOX, and the elastin degradation products ELM and ELM-2?

BACKGROUND: Diabetes mellitus type 2 (T2DM) is a significant risk factor for the development of cardiovascular diseases (CVDs). In a previous microarray study of internal mammary arteries from patients with and without T2DM, we observed several elastin-related genes with altered mRNA-expression in diabetic patients, namely matrix metalloproteinase 2 (MMP-2), lysyl oxidase (LOX) and elastin itself. In this study we investigate whether the serum concentrations of elastin-related proteins correlate to signs of CVD in patients with T2DM. METHODS: Blood samples from 302 type 2 diabetic patients were analysed for MMP-2, LOX, and the elastin degradation products ELM and ELM2. The results were investigated for correlations to signs of CVD in different vascular territories, as determined by myocardial perfusion scintigraphy, carotid artery thickness and ankle-brachial blood pressure index. RESULTS: T2DM patients with peripheral arterial disease (low ankle-brachial index) (PAD) display higher levels of MMP-2 and ELM compared to patients without PAD. However, none of the proteins or degradation products correlated with myocardial ischemia or a combined measure of CVD-signs, including myocardial ischemia, increased carotid thickness and decreased ankle-brachial blood pressure. CONCLUSIONS: Our results suggest that the diabetic environment affects the circulating amounts of MMP-2 and ELM in patients with PAD. However, the same connection could not be seen in diabetic patients with CVD broadly identified in three vascular territories. LOX and ELM-2 did not correlate to any type of CVD. Overall, serum levels of elastin-related molecules are only remotely related to CVD in type 2 diabetes.

Antenatal glucocorticoids and neonatal inflammation-associated proteins.

BACKGROUND: To date, studies of the relationship between antenatal glucocorticoids (AGC) and neonatal inflammation in preterm newborns have been largely limited to umbilical cord blood specimens. AIM: To explore the association between exposure to antenatal glucocorticoids and concentrations of inflammation-related proteins in whole blood collected from very preterm newborns at multiple times during the first postnatal month. METHODS: We measured the protein concentrations on postnatal day 1 (N=1118), day 7 (N=1138), day 14 (N=1030), day 21 (N=936) and day 28 (N=877) from infants born before the 28th week of gestation and explored the relationship between antenatal steroid receipt and protein concentrations in the highest and lowest quartiles. The creation of multinomial logistic regression models (adjusted for potential confounders) allowed us calculate odds ratios and 95% confidence intervals. RESULTS: Twenty of 420 assessments [21 (proteins)×2 (exposure levels: partial and full)×2 (quartile levels: top and bottom)×5 (days)] were statistically significant without any cohesive pattern. CONCLUSION: Among infants born before 28 weeks of gestational age, neither full, nor partial courses of antenatal glucocorticoids have a sustained anti-inflammatory effect.

Monoclonal antibodies specific to heat-treated porcine blood.

BACKGROUND: Porcine blood is potentially being utilized in food as a binder, gelling agent, emulsifier or colorant. However, for certain communities, the usage of animal blood in food is strictly prohibited owing to religious concerns and health reasons. This study reports the development of monoclonal antibodies (MAbs) against heat-treated soluble proteins (HSPs) of autoclaved porcine blood; characterization of MAbs against blood, non-blood and plasma from different animal species using qualitative indirect non-competitive enzyme-linked immunosorbent assay (ELISA); and immunoblotting of antigenic components in HSPs of porcine blood. RESULTS: Fifteen MAbs are specific to heat-treated and raw porcine blood and not cross-reacted with other animal blood and non-blood proteins (meat and non-meat). Twelve MAbs are specific to porcine plasma, while three MAbs specific to porcine plasma are cross-reacted with chicken plasma. Immunoblotting revealed antigenic protein bands (∼60, ∼85-100 and ∼250 kDa) in porcine blood and plasma recognized by the MAbs. CONCLUSION: Selection of MAbs that recognized 60 kDa HSPs of porcine blood and plasma as novel monoclonal antibodies would be useful for detection of porcine plasma in processed food using the immunoassay method. © 2015 Society of Chemical Industry.

Probing Bio-Nano Interactions between Blood Proteins and Monolayer-Stabilized Graphene Sheets.

Meeting proteins is regarded as the starting event for nanostructures to enter biological systems. Understanding their interactions is thus essential for a newly emerging field, nanomedicine. Chemically converted graphene (CCG) is a wonderful two-dimensional (2D) material for nanomedicine, but its stability in biological environments is limited. Systematic probing on the binding of proteins to CCG is currently lacking. Herein, we report a comprehensive study on the interactions between blood proteins and stabilized CCG (sCCG). CCG nanosheets are functionalized by monolayers of perylene leading to significant improvement in their resistance to electrolyte salts and long-term stability, but retain their core structural characteristics. Five types of model human blood proteins including human fibrinogen, γ-globulin, bovine serum albumin (BSA), insulin, and histone are tested. The main driving forces for blood protein binding involve the π-π interacations between the π-plane of sCCG and surface aromatic amonic acid (sAA) residues of proteins. Several key binding parameters including the binding amount, Hill coefficient, and binding constant are determined. Through a detailed analysis of key controlling factors, we conclude that the protein binding to sCCG is determined mainly by the protein size, the number, and the density of the sAA.