Plasma Protein Fractions Research Articles

DIGESTIVE AND METABOLIC RESPONSES TOWARDS DIET OPTIMIZATION IN LABEO ROHITA

Digestive and metabolic responses of Labeo rohita (av. wt. 1.07–1.14 g) to different dietary crude protein (CP) levels ( 25, 30, 35, 40 and 45%) were studied to optimize a practical diet formulation for this species. Soybean meal and fish meal were served as the main protein sources, whereas corn flour and rice polish as major carbohydrate sources. After 45 days of feeding, trypsin and chymotrypsin activities were lower in 25% CP group. Alkaline phosphatase and trypsin activities were maximized (p<0.05) in 35% CP group. Amylase activity was not influenced by the diet composition. Hexokinase (HK) activity in the muscle of fish fed 25 to 35% CP was significantly higher (p<0.05) than the other two groups. Liver also recorded a higher HK activity in low protein fed groups. Similarly, muscle and liver pyruvate kinase activities were significantly higher in 25 and 30% CP fed groups. Malate dehydrogenase activity in muscle was highest at 30% CP group. Muscle aspartate amino-transferase activity was found to be higher in low protein fed groups ( 25 and 30%). Metabolites showed significant change corresponding to the dietary composition. Glucose and glycogen levels matched well with the dietary carbohydrate levels. Muscle and plasma pyruvic acid contents increased as the dietary protein increased, whereas liver pyruvic acid showed opposite trend. Protein fractions in plasma (total protein, albumin and globulin) showed maximum values in 30% CP group. Key words: Crude protein, digestive enzymes, metabolic enzymes, metabolites, Labeo rohita

Studies on the kinetics, metabolism and re-utilisation after intra-articular administration of hyaluronan to rabbits.

The pharmacokinetics of hyaluronan (HA) (CAS 9004-61-9) was measured after intra-articular (i.a.) injection of 1 mg/kg of 14C-glucose-labelled HA (14C-HA) (i.e. 2,000 kDa) into the knee joint cavity of rabbits. The movement of HA from the cavity into the systemic circulation was assessed by measuring its concentration in blood and its residue in the cavity. I.a. HA moved into, and disappeared from, the bloodstream at a first-order rate. Synovial fluid, synovial membrane and articular cartilage specimens were taken 24 and 72 h post-injection, and the molecular weight (MW) of HA fractions were measured by using gel filtration chromatography (GFC) using radioactive fractions of known molecular sizes (i.e. 2,000 kDa, 300 kDa and 50 kDa). The radioactivity distribution of synovial fluid had a peak at a 2,000 kDa-equivalent fraction at both 24 and 72 h post-injection, while synovial membrane had a peak at a 300 kDa-equivalent fraction 24 h post-injection and at a 50 kDa-equivalent fraction 72 h post-injection. The radioactivity distribution pattern of articular cartilage showed peaks corresponding to 2,000-300 kDa 24 h post-injection and to 50 kDa 72 h post-injection. The in vivo re-utilisation of i.a. HA was investigated by assessing radioactivity in the acid-soluble, lipid and protein fractions of plasma, blood cells and liver 72 h post-injection. Results show that HA was broken down into C1 units (carbon cycle) before being re-used as an in vivo constituent in the body.

Human albumin solution for resuscitation and volume expansion in critically ill patients

Human albumin solutions are used for a range of medical and surgical problems. Licensed indications are the emergency treatment of shock and other conditions where restoration of blood volume is urgent, such as in burns and hypoproteinaemia. Human albumin solutions are more expensive than other colloids and crystalloids. To quantify the effect on mortality of human albumin and plasma protein fraction (PPF) administration in the management of critically ill patients. We searched the Cochrane Injuries Group Specialised Register (searched 31 May 2011), the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library 2011, Issue 2), MEDLINE (Ovid) (1948 to week 3 May 2011), EMBASE (Ovid) (1980 to Week 21 2011), CINAHL (EBSCO) (1982 to May 2011), ISI Web of Science: Science Citation Index Expanded (SCI-EXPANDED) (1970 to May 2011), ISI Web of Science: Conference Proceedings Citation Index - Science (CPCI-S) (1990 to May 2011), PubMed (www.ncbi.nlm.nih.gov/sites/entrez/) (searched 10 June 2011, limit: last 60 days). Reference lists of trials and review articles were checked, and authors of identified trials were contacted. Randomised controlled trials comparing albumin or PPF with no albumin or PPF or with a crystalloid solution in critically ill patients with hypovolaemia, burns or hypoalbuminaemia. We collected data on the participants, albumin solution used, mortality at the end of follow up, and quality of allocation concealment. Analysis was stratified according to patient type. We found 38 trials meeting the inclusion criteria and reporting death as an outcome. There were 1,958 deaths among 10,842 trial participants.For hypovolaemia, the relative risk of death following albumin administration was 1.02 (95% confidence interval (CI) 0.92 to 1.13). This estimate was heavily influenced by the results of the SAFE trial, which contributed 75.2% of the information (based on the weights in the meta-analysis). For burns, the relative risk was 2.93 (95% CI 1.28 to 6.72) and for hypoalbuminaemia the relative risk was 1.26 (95% CI 0.84 to 1.88). There was no substantial heterogeneity between the trials in the various categories (Chi(2) = 26.66, df = 31, P = 0.69). The pooled relative risk of death with albumin administration was 1.05 (95% CI 0.95 to 1.16). For patients with hypovolaemia, there is no evidence that albumin reduces mortality when compared with cheaper alternatives such as saline. There is no evidence that albumin reduces mortality in critically ill patients with burns and hypoalbuminaemia. The possibility that there may be highly selected populations of critically ill patients in which albumin may be indicated remains open to question. However, in view of the absence of evidence of a mortality benefit from albumin and the increased cost of albumin compared to alternatives such as saline, it would seem reasonable that albumin should only be used within the context of well concealed and adequately powered randomised controlled trials.

Reference intervals for biochemical analytes in serum and heparinized plasma and serum protein fractions in adult alpacas (Vicugna pacos)

Species-specific reference intervals for laboratory tests are required for diagnosis and management of disease. Few reports of reference intervals for biochemical analytes and protein fractions are available for alpacas. The aims of this study were to establish reference intervals for biochemical analytes measured in both serum and plasma and in serum protein fractions, and to determine the influence of herd and sex on test results in a population of alpacas. Blood was collected from 74 healthy male and female adult alpacas (Vicugna pacos) from 5 herds into tubes with no anticoagulant or with sodium heparin and analyzed within 4 hours. Biochemical analytes and ionized calcium were measured using a Hitachi P modular automated chemistry analyzer and an ABL-800 Flex blood-gas analyzer, respectively, and protein fractions were measured by agarose gel electrophoresis of serum. Nonparametric statistical methods were used to determine reference intervals, results obtained from serum and plasma were compared, and effects of herd and sex were examined. Serum and plasma samples from 71 and 74 alpacas, respectively, were used to establish reference intervals for serum and plasma biochemical analytes. Intervals were similar, although clinically relevant differences between creatine kinase activity and phosphate concentration were found in individual animals. Serum proteins from 60 alpacas were analyzed by electrophoresis. There were significant herd- and sex-associated differences in some biochemical analytes and protein fractions; however, most had minimal impact on reference interval determination, with the exception of herd-associated effects on concentrations of urea nitrogen, ionized calcium, and bile acids and transferrin saturation. Serum and plasma reference intervals are interchangeable; however, consistency of sample type is imperative when performing serial testing. Use of laboratory- and instrument-specific reference intervals is optimal; however, intervals reported here may be used as a guide for interpreting laboratory results from alpacas, especially when test methods are the same.

Impact of Intensive Physical Activity on Selenium Status

Various biomarkers were used to assess selenium (Se) status during 3 months of basic military training in a group of recruits. Samples of whole blood and plasma or serum were taken from a group of conscripts three times: at the beginning (n = 15), in the middle immediately after a severely stressful physical activity (n = 15) and at the end of military training (n = 13). Selenium was determined in diet samples, blood, plasma and plasma protein fractions as selenoprotein P (SelP) and glutathione peroxidase (eGPx). Selenium was determined by hydride generation-atomic fluorescence spectrometry and fractionation of proteins performed by affinity chromatography. Total serum glutathione, erythrocyte and serum GPx activity were followed as well. The average Se intake was calculated according to meal consumption questionnaires and Se determined in composite diet samples, giving an assessed intake of 48 ± 10 μg/day. At all three samplings, the average blood Se concentration was within the framework of adequate supply (87 ± 12, 94 ± 15 and 80 ± 13 ng/g). Plasma Se was between 70 and 80 ng/g (71 ± 10, 79 ± 9 and 76 ± 10 ng/g), which is believed to enable the full expression of plasma GPx. The average shares of plasma Se proteins were 61 ± 6%, 58 ± 6% and 54 ± 9% for SelP and 27 ± 4%, 34 ± 7% and 29 ± 5% for GPx. Although the observed tendency of the increases of serum and erythrocyte GPx activities at the second and third samplings with respect to the first was statistically insignificant, it is still indicative of some protection against oxidative stress, while the decreasing SelP levels during training suggest a slowly decreasing biologically active selenium pool.

Use of plasma protein fraction in patients with septic shock admitted to the ICU

Certain colloids like albumin and plasma protein fraction (PPF) have been derived from human plasma and they are used as plasma expanders to treat patients with shock. PPF, which more closely resembles plasma in its constituents, contains albumin plus α and β globulins. We conducted this study to assess the effect of PPF on need for vasopressors, organ support and ICU mortality in patients with septic shock.

Differences in Protein Fractions of Avian Plasma Among Three Commercial Electrophoresis Systems

Previous studies have defined the presence of 6 protein fractions in plasma from many psittaciform species. Additionally, extensive reference intervals have been published for many of these species with the Beckman Paragon electrophoresis system, which had been commonly used in clinical laboratories to analyze the protein fractions of avian plasma. In mid-2009, Beckman discontinued the Paragon product line, leaving 2 primary alternative systems: Helena and Sebia. To compare electrophoresis results from the 3 commercial protein electrophoresis systems, specimens from 40 African grey parrots (Psittacus erithacus) were analyzed with the electrophoresis systems from Beckman, Helena, and Sebia. Marked differences in fraction migration were found between the Beckman/Helena and Sebia systems, which manifested as a large decrease in prealbumin and an increase in alpha1 globulins in the latter system. Both proportional and constant errors were observed among the fraction quantitation data of both the Helena and Sebia systems compared with the Beckman system. Based on Bland-Altman plot data and imprecision studies, the Helena system appears more similar with the Beckman system, although neither the Helena nor the Sebia systems are identical to the Beckman system. Because of the differences in electrophoresis methods, clinicians should be careful to consistently use particular clinical laboratories. For best application, reference intervals should be established based on both species and electrophoresis system.

Zinc-binding proteins from boar seminal plasma -- isolation, biochemical characteristics and influence on spermatozoa stored at 4°C.

Affinity chromatography on Chelating Sepharose Fast Flow Gel-Zn(2+) was used for fractionation of boar seminal plasma proteins. Approximately 30% of total boar seminal plasma proteins showed affinity for zinc ions (ZnBP fraction). Native electrophoresis (PAGE) of ZnBP revealed six protein fractions which separated into 27 bands under denaturing conditions (SDS/PAGE). Two-dimensional electrophoresis (2D PAGE) showed 148 polypeptides with isoelectric points mostly in the basic and neutral pH range. The zinc-binding proteins comprise mainly 10-20 kDa polypeptides which are probably members of the spermadhesin family. ZnBP present in the incubation mixture of spermatozoa stored for 1 or 24 h at 4 °C allowed preservation of a higher percentage of cells exhibiting linear motility in comparison to a control sample stored in PBS. Presented results indicate that proteins binding Zn(2+) ions have a shielding effect on the sperm plasma membrane and acrosome of spermatozoa, protecting these structures against consequences of cold shock.

Relationship between late embryonic mortality and the increase in plasma advanced oxidised protein products (AOPP) in dairy cows

The involvement of protein oxidation in embryonic mortality (EM) has been poorly investigated in cows. Advanced oxidation protein products (AOPP) are markers of protein oxidation generated by activated neutrophils and involved in inflammation. The aim of this work was to study AOPP in cow plasma and their relationship with late EM. The outcomes of 158 artificial inseminations (AI) were examined in 72 cows, which were classified ex post on the basis of blood progesterone and pregnancy-associated glycoprotein concentrations and clinical confirmation of pregnancy into the following categories: (1) positive (AI+, resulted in pregnancy, n=58), (2) negative (AI-, did not result in pregnancy, n=86) and (3) embryonic mortality (EM, n=14). Plasma protein fractions, malondialdehyde (MDA), total glutathione and AOPP were measured at AI (Day 0) and on Days 15, 28, 35, 45 and 60. MDA was significantly higher in EM than AI+ and AI- animals on Day 45, and than AI+ animals on Day 60 (P<0.05). Mean plasma AOPP concentrations were significantly higher in the EM group (P<0.01) and the ratio of AOPP:albumin was significantly higher in the EM group on Days 15, 28, 45 and 60 (P<0.05). Based on the temporal pattern of the AOPP:albumin ratio, we propose that oxidative stress is implicated in and may possibly be a cause of EM.

Peritoneovenous shunt for intractable ascites due to hepatic lymphorrhea after hepatectomy

A peritoneovenous shunt has become one of the most efficient procedures for intractable ascites due to liver cirrhosis. A case of intractable ascites due to hepatic lymphorrhea after hepatectomy for hepatocellular carcinoma that was successfully treated by the placement of a peritoneovenous shunt is presented. A 72-year-old Japanese man underwent partial resection of the liver for hepatocellular carcinoma associated with hepatitis C viral infection. After hepatectomy, a considerable amount of ascites ranging from 800-4600 mL per day persisted despite conservative therapy, including numerous infusions of albumin and plasma protein fraction and administration of diuretics. Since the patient's general condition deteriorated, based on the diagnosis of intractable hepatic lymphorrhea, a subcutaneous peritoneovenous shunt was inserted. The patient's postoperative course was uneventful and the ascites decreased rapidly, with serum total protein and albumin levels and hepatic function improving accordingly. For intractable ascites due to hepatic lymphorrhea after hepatectomy, we recommend the placement of a peritoneovenous shunt as a procedure that can provide immediate effectiveness without increased surgical risk.

Changes of doxorubicin distribution in blood and plasma after its inclusion into nanophospholipd formulation

The drug composition based on the plant phospholipids and the antitumor drug doxorubicin (particle size <30 nm) was obtained using original technology elaborated in the Institute of Biomedical Chemistry (Russian Academy of Medical Sciences). In in vitro experiments demonstrated decreased drug association with blood cells for this nanophospholipid form as compared with free doxorubicin. This was accompanied by a with corresponding increase in its plasma level ans also by drug redistribution from plasma protein fraction to high density lipoproteins. Significance of these changes for doxorubicin biodistributon and antitumor activity is discussed.

Improved particle-packed HPLC/MS microchips for proteomic analysis

The influence of packing process parameters (packing pressure, application of ultrasound) and the stationary phase particle size (3.5 and 5 μm) on the chromatographic performance of HPLC/MS chips was systematically investigated for proteomic samples. First, reproducibility and detection limits of the separation were evaluated with a low-complexity sample of tryptic BSA peptides. The influence of adsorbent packing quality on protein identification was then tested with a typical proteomics sample of high complexity, a human plasma protein fraction (Cohn fraction IV-4). All HPLC/MS chips provided highly reproducible separations of these proteomic samples, but improved packing conditions and smaller particle sizes resulted in chromatograms with narrower peaks and correspondingly higher signal intensities. Improved separation performance increased the peak capacity, the number of identified peptides, and thus the sequence coverage in the proteomic samples, particularly for low sample amounts.

Depletion of Abundant Plasma Proteins and Limitations of Plasma Proteomics

Immunoaffinity depletion with antibodies to the top 7 or top 14 high-abundance plasma proteins is used to enhance detection of lower abundance proteins in both shotgun and targeted proteomic analyses. We evaluated the effects of top 7/top 14 immunodepletion on the shotgun proteomic analysis of human plasma. Our goal was to evaluate the impact of immunodepletion on detection of proteins across detectable ranges of abundance. The depletion columns afforded highly repeatable and efficient plasma protein fractionation. Relatively few nontargeted proteins were captured by the depletion columns. Analyses of unfractionated and immunodepleted plasma by peptide isoelectric focusing (IEF), followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), demonstrated enrichment of nontargeted plasma proteins by an average of 4-fold, as assessed by MS/MS spectral counting. Either top 7 or top 14 immunodepletion resulted in a 25% increase in identified proteins compared to unfractionated plasma. Although 23 low-abundance (<10 ng mL(-1)) plasma proteins were detected, they accounted for only 5-6% of total protein identifications in immunodepleted plasma. In both unfractionated and immunodepleted plasma, the 50 most abundant plasma proteins accounted for 90% of cumulative spectral counts and precursor ion intensities, leaving little capacity to sample lower abundance proteins. Untargeted proteomic analyses using current LC-MS/MS platforms-even with immunodepletion-cannot be expected to efficiently discover low-abundance, disease-specific biomarkers in plasma.

Oral plasma proteins attenuate gut inflammatory effects induced by S. aureus enterotoxin B challenge in rats

In previous studies we have shown that dietary supplementation with spray-dried porcine plasma proteins (SDP) attenuates the effects of the S. aureus enterotoxin B (SEB) on activation of gut-associated lymphoid tissue (GALT) and increased intestinal permeability. We have now studied if the effects of plasma supplementation correlated with mucosal cytokine release and defensin expression. Wistar–Lewis rats were fed diets supplemented with SDP (8% w/w), with a plasma protein fraction containing 50% Ig (PPF; 1.5% w/w) or with milk proteins (Control diet), Animals were fed experimental diets from weaning (day 21) until day 35 after birth, and on days 32 and 35, were administered SEB (i.p. 0.5 mg/kg) to induce a mild intestinal inflammation. Experimental groups were Control, SEB, SEB-SDP and SEB-PPF. Cytokines were determined by a cytometric bead array assay, LTB 4 by ELISA and iNOS and defensins by real time PCR, all at 6 h after SEB administration. Results showed that SEB significantly increased mucosal IFN-γ, TNF-α, IL-6 and LTB 4 concentration (by 110%, 20%, 30% and 110%, respectively; all P < 0.05). SDP and PPF partially attenuated these effects. SEB increased mucosal iNOS expression by 22% (P < 0.05) and both plasma protein supplements prevented SEB effects on iNOS expression (both P < 0.05). SEB administration also reduced the expression of β-defensin 1 and cryptdin 4 about 10% (P < 0.05) and both supplements significantly prevented these effects (P < 0.05). SDP increased the expression of mucosal IL-10 supporting the view that this anti-inflammatory cytokine participates in the mechanism of action of dietary plasma proteins during intestinal inflammation.

Free vs Total Pregnancy-Associated Plasma Protein A (PAPP-A) as a Predictor of 1-Year Outcome in Patients Presenting with Non–ST-Elevation Acute Coronary Syndrome

The free fraction of pregnancy-associated plasma protein A (FPAPP-A) was found to be the PAPP-A form released to the circulation in acute coronary syndrome (ACS). We estimated the prognostic value of FPAPP-A vs total PAPP-A (TPAPP-A) concentrations in forecasting death and nonfatal myocardial infarction (combined endpoint) in patients with non-ST-elevation ACS. We recruited 267 patients hospitalized for symptoms consistent with non-ST-elevation ACS and followed them for 12 months. FPAPP-A, TPAPP-A, C-reactive protein (CRP), and cardiac troponin I (cTnI) were measured at admission; cTnI was also measured at 6-12 h and 24 h. Because of the recently shown interaction between PAPP-A and heparin, we excluded patients treated with any heparin preparations before the admission blood sampling. During the follow-up, 57 (21.3%) patients met the endpoint (22 deaths and 35 nonfatal myocardial infarctions). According to FPAPP-A (<1.27, 1.27-1.74, >1.74 mIU/L) and TPAPP-A (<1.98, 1.98-2.99, >2.99 mIU/L) tertiles, this endpoint was met by 12 (13.5%), 18 (20.2%), 27 (30.3%) (P = 0.02), and 17 (19.1%), 17 (19.1%), 23 (25.8%) (P = 0.54) patients, respectively. After adjusting for age, sex, diabetes, previous myocardial infarction, and ischemic electrocardiogram (ECG) findings, FPAPP-A >1.74 mIU/L [risk ratio (RR) 2.0; 95% CI 1.0-4.1, P = 0.053), increased cTnI, and CRP >/=2.0 mg/L were independent predictors of an endpoint. The prognostic performance of TPAPP-A was inferior to that of FPAPP-A. FPAPP-A seems to be superior as a prognostic marker compared to TPAPP-A, giving independent and additive prognostic information when measured at the time of admission in patients hospitalized for non-ST-elevation ACS.

Conventional blood banking and blood component storage regulation: opportunities for improvement.

Blood banking is a medical logistic activity. It attempts to bring the potentially life-saving benefits of transfusion to the patients who need them by making blood components available, safe, effective and cheap1. Blood banks try to maximize delivering getting blood from the right donors to the right patients in a timely manner. The easiest way to assure the timely availability of blood is to have an appropriate inventory on the shelf at all times. Standards for blood banking have evolved in response to problems observed in the past2. Donors need to be free of syphilis, hepatitis, and human immunodeficiency virus (HIV) and from a host of other diseases as well. Methods for cleaning the arms of donors should work. Blood bags should contain the appropriate solutions and be sterile. Systems for the identification of donors and patients, for the determination of antigens on their blood cells and the antibodies in their sera, and for the procedures and processes used to gather and maintain this information should be robust. Regulatory agencies, like the United States (U.S.) Food and Drug Administration (FDA), are charged to assure that blood products are safe and effective3. They attempt to assure safety by enforcing the standards noted above. They attempt to assure effectiveness by demanding demonstrations that a reasonable fraction of red cells, platelets and plasma proteins survive after storage before licensing new blood storage systems or blood modifying products like leukocyte reduction filters4,5. Yet the reasonable fraction for red cells is 75%, for platelets is 67%, and for plasma proteins is 80%. Standards, such as insisting that every unit of blood be tested for HIV, work only if the licensed tests detect the circulating viruses, a moving target. Better understanding of the basic biology and better tests on which to base better standards are needed. If we understood the changes that occur with the storage of red cells, platelets and plasma better, we could both design better storage systems and regulate storage more effectively. If we knew more about the viruses and bacteria and the cytokines and blood breakdown products that threaten blood safety we could institute controls to further improve blood safety. This paper will explore some specific examples.

Non-invasive fetal RHD genotyping in the first trimester of pregnancy

Hemolytic disease of the fetus and newborn (HDN) is caused primarily by feto-maternal RhD incompatibility. Although all RhD negative pregnant women undergo routine antenatal RhD prophylaxis at 28 weeks of gestation, and following delivery if the newborn is RhD positive, HDN has not been eradicated. Here, we investigated fetal Rhesus D (RHD) genotype in maternal plasma during the first trimester of pregnancy in our area. Plasma samples were obtained from 111 RhD negative pregnant women, between 9 and 13 weeks of gestation. DNA from maternal plasma containing cell-free fetal DNA (cffDNA) was analyzed by quantitative PCR (qPCR) to detect RHD exons 5 and 7. A beta-globin (HBB) sequence was quantified to estimate total DNA concentration. qPCR results were compared with newborn RhD determined in cord blood serum. The influence of several gestational parameters on DNA concentration was also analyzed. The specificity and sensitivity of the assay was 93% and 100%, respectively, with 97% diagnostic accuracy. Cell-free DNA concentrations during the first trimester of pregnancy were not affected by the gestational parameters studied (free-beta fraction of human chorionic gonadotropin and pregnancy-associated plasma protein A concentrations, fetal sex, materno-fetal ABO blood group incompatibility, maternal weight and gestational age). Non-invasive fetal RHD genotyping during the first trimester of pregnancy can be determined with a high specificity, thus representing a valuable tool for improving the management of RhD negative pregnant women. As a high percentage of pregnant women participate in the routine first trimester combined screening program for aneuploidies, the fetal RHD study could be of immediate implementation, since the same blood collection could be used.

Dendritic/pancreatic carcinoma fusions for clinical use: Comparative functional analysis of healthy- versus patient-derived fusions

Fetal calf serum (FCS)-independent pancreatic cancer cells were established in plasma protein fraction (PPF)-supplemented medium that is an agent of good manufacturing practice (GMP) grade. Dendritic cells (DCs) were activated with the Toll-like receptor agonist, penicillin-inactivated Streptococcus pyogenes (OK-432) that is also a GMP grade agent. Therefore, sufficient amounts of FCS-independent fusions were successfully generated with decreased potential hazards of FCS. The FCS-independent fusions expressed tumor-associated antigens, HLA-DR, costimulatory molecules, IL-12, and IL-10. Stimulation of T cells with fusions from healthy donors resulted in proliferation of T cells with high expression levels of perforin/granzyme B and IFN-gamma and efficient induction of antigen-specific cytotoxic T lymphocytes (CTLs). Selection and expansion of T-cell clones were confirmed by TCR Vbeta analysis. However, fusions from patients with metastatic pancreatic cancer induced increased expression levels of TGF-beta1 in CD4+ CD25high T cells and low levels of CTLs with decreased IFN-gamma production.

Application of displacement chromatography for the proteome analysis of a human plasma protein fraction

It was the aim of this study to compare the performance of displacement chromatography with gradient elution chromatography both applied as the cation-exchange separation step for a proteome analysis in a bottom-up approach using multidimensional chromatography for the separation of tryptic peptides prior to their mass spectrometric analysis. The tryptic digest of the human Cohn fraction IV-4 served as a sample. For both chromatography modes commonly used operating parameters were chosen thus ensuring optimal separation results of equal sample amounts for each mode. All resulting fractions were analyzed with an HPLC-chip–LC–MS system. The eluate of the HPLC-chip column was ionized by electrospray ionization (ESI) and analyzed with an ion-trap mass spectrometer. For guaranteeing high confidence concerning the identity of the peptides, the mass spectrometric data were processed by different bioinformatic tools applying stringent criteria. By the displacement approach the total amount of identified proteins (78) was significantly higher than in the gradient mode (58). The results showed that displacement chromatography is a well suited alternative in comparison to gradient elution separation for analysis of proteomes via the bottom-up approach applying multidimensional chromatography, especially in those cases when larger quantities of proteins are available.

Hepcidin, the hormone of iron metabolism, is bound specifically to α-2-macroglobulin in blood

AbstractHepcidin is a major regulator of iron metabolism. Hepcidin-based therapeutics/diagnostics could play roles in hematology in the future, and thus, hepcidin transport is crucial to understand. In this study, we identify α2-macroglobulin (α2-M) as the specific hepcidin-binding molecule in blood. Interaction of 125I-hepcidin with α2-M was identified using fractionation of plasma proteins followed by native gradient polyacrylamide gel electrophoresis and mass spectrometry. Hepcidin binding to nonactivated α2-M displays high affinity (Kd 177 ± 27 nM), whereas hepcidin binding to albumin was nonspecific and displayed nonsaturable kinetics. Surprisingly, the interaction of hepcidin with activated α2-M exhibited a classical sigmoidal binding curve demonstrating cooperative binding of 4 high-affinity (Kd 0.3 μM) hepcidin-binding sites. This property probably enables efficient sequestration of hepcidin and its subsequent release or inactivation that may be important for its effector functions. Because α2-M rapidly targets ligands to cells via receptor-mediated endocytosis, the binding of hepcidin to α2-M may influence its functions. In fact, the α2-M–hepcidin complex decreased ferroportin expression in J774 cells more effectively than hepcidin alone. The demonstration that α2-M is the hepcidin transporter could lead to better understanding of hepcidin physiology, methods for its sensitive measurement and the development of novel drugs for the treatment of iron-related diseases.