Plasma Protein Fractions Research Articles

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.

Protecting the blood supply from emerging pathogens: The role of pathogen inactivation

As a consequence of the many blood-safety interventions introduced since the mid-1980s, the major causes of transfusion-associated mortality have shifted from being mainly due to transfusion-transmitted infections (TTIs) to being mainly due to non-infectious serious events such as TRALI, hemolytic reactions, transfusion overload, and graft versus host disease. Thus, TTIs now account for only 10 to 15% of all transfusion associated mortalities! Relevantly, manufacturers of purified plasma protein fractions have, over the same time period, shown that pathogen inactivation technologies can be successfully implemented resulting in little or no transmission of HIV, HCV or HBV since the late 1980s. These technologies, however, cannot be applied to cellular blood components. Thus, new technologies have evolved over the past decade to treat cellular blood components as well as plasma. These technologies, particularly those involving plasma and platelets, have begun to be used in Europe and this proactive paradigm has evolved to become a potential pre-emptive approach for ridding the blood supply of most TTIs (virus, bacteria, and protozoa). However, in order for pathogen inactivation technologies to become widely accepted, they must be shown to be both cost effective and not associated with new risks to recipients!

New methods of plasma fractionation – a presentation of the ‘mini‐pool’ fractionation procedure developed in Egypt

Background Availability of safe and affordable human plasma‐derived products (factor VIII, prothrombin complex concentrate/FIX, albumin and intravenous immunoglobulin) is a main concern of healthcare providers, particularly in resource‐limited countries. Several mechanisms may be used at national levels to secure the supply of these products, including import of finished products, contract fractionation and/or domestic fractionation. Contract fractionation may be considered only when there is a sufficient and steady amount of plasma (typically well over 10 000 l/year) complying with the international quality and regulatory requirements for fractionation, prepared following good manufacturing practice (GMP) principles, and ideally generated by a nationally coordinated blood transfusion organization. Fractionated products obtained by contract fractionation are not necessarily cheaper than imported ones. A domestic fractionation facility requires the guarantee to have access to a larger volume of plasma (typically over 100 000–200 000 l/year) that should also strictly comply with international quality and regulatory requirements. This option implies the construction, qualification and validation of a very high cost GMP pharmaceutical fractionation facility, use of state‐of‐the‐art technologies, and availability and training of highly qualified staff. The local market should have the capacity to use at least three out of the main four products to ensure the financial viability of the project. Due to these constraints, both contract fractionation and domestic fractionation may not currently be feasible for a number of resource‐limited countries.Objectives As an initial alternative to the conventional approaches to secure the supply of plasma products, our group started in 2003 to develop a new concept for the preparation of plasma protein components at a ‘mini‐pool’ fractionation scale (MPFS).Methods The main feature relies on the application of a single‐use sterile bag system process that can be implemented by blood establishments or a national service centre. The technology is straightforward and uses equipment that is largely available at blood establishments. The current pool size of 5–10 l of plasma can be largely processed, in an enclosed single‐use bag system, without the need of expensive fixed large‐scale equipment. A validated universal virus inactivation technology (solvent‐detergent) has been successfully integrated into this MPFS process, using such single‐use disposable bags.Results Mini‐pool, in‐bag, virus inactivation of cryoprecipitate, fresh‐frozen plasma and cryo‐poor plasma have been successfully developed and the medical device will soon be marketed. A factor VIII‐enriched preparation, also containing von Willebrand factor, has been obtained at a yield of about 300 IU/l, a concentration of 20–25 IU/ml, and a specific activity of 5–10 IU/mg. A chromatographic separation process of a PCC component from cryo‐poor plasma at a yield of 300 IU factor IX/l, containing factors II, VII and X was also developed. Work on separation of albumin‐ and Intravenous immunoglobulin‐enriched preparations is in progress.Conclusion This MPFS offers numerous advantages, among which is avoiding the need of an expensive GMP facility. The plasma mini‐pool size (5–10 l) gives flexibility of production of different combination of plasma component fractions. The concept that should be applied following GMP represents a gateway for resource‐limited countries to make use of their domestic plasma to improve the supply of plasma protein fractions and the standard of care of patients. The technology can also be used for preparation of safe plasma products for rare bleeding disorders.

Accumulation of 3H-1-lysine by brain and plasma in male and female rats treated acutely wigh morphine sulfate.

Adult male and female Wistar rats were injected with a single dose of 40 mg/kg of morphine sulfate via indwelling intrajugular cannulas and killed two hours later. 3H-1-lysine was injected at various time intervals before the death of the animals. The distribution of the labelled amino acid was then determined in the free amino acid and lipid-nucleoprotein-extracted protein fractions of plasma and cerebral grey matter. The accumulation of 3H-activity was also determined in various areas of the brain, dorsal root ganglia, pectoral muscle and liver. Morphine-treatment produced an elevation of free 3H-lysine levels in plasma and cerebral grey matter and a decrease in incorporation into protein. A significant sex difference was noted, such that in control males the plasma free 3H-lysine and protein fractions were higher than in females. In the morphine-treated group the values obtained from males were lower than for females. In cerebral grey matter, the free 3H-lysine was significantly lower in control and morphine-treated males than in females. In the protein fraction, such sex differences varied with the time elapsed after injection of amino acid and did not clearly favor either sex. The accumulation of 3H-activity in tissue blocks taken from various regions of CNS was unaffected by morphine-treatment in subcortical components. The values were generally higher in females. 3H-accumulation was significantly altered in cortical components in the male group. In cerebral white matter, the morphine-treated males showed significant elevations of accumulation. Females showed an elevation of 3H-accumulation in the morphine-treated group only when the data was subjected to a group comparison. In the peripheral nervous system, 3H-accumulation was significantly depressed by morphine-treatment only in males. The female values were again significantly higher than those from the males. The male liver demonstrated a significant depression in accumulation of 3H-activity in the morphine-treated group. Liver tissue from females was less responsive and had higher uptakes than in males. Skeletal muscle showed no alteration in 3H-accumulation after morphine treatment in either sex.

Seminal plasma proteins protect flow-sorted ram spermatozoa from freeze - thaw damage

Seminal plasma improves the functional integrity of compromised ram spermatozoa but has been reported to be toxic to sorted spermatozoa. The present study attempted to clarify this paradoxical effect and improve the functional integrity of spermatozoa following sorting and cryopreservation. The in vitro function of sorted spermatozoa (motility characteristics and membrane integrity) was examined after supplementation with differing concentrations and protein fractions of seminal plasma at various stages of the sorting and freezing process. For all experiments, spermatozoa (two males, n=four ejaculates per male) were processed through a high-speed flow cytometer before cryopreservation, thawing and incubation for 6 h (37 degrees C). Supplementation of crude seminal plasma (CP), its low molecular weight fraction (LP; <10 kDa) or protein-rich fraction (SPP; >10 kDa), immediately before freezing improved the functional integrity of sorted spermatozoa compared with no supplementation (control), whereas supplementation after thawing had no effect for CP and LP. The protective effect of seminal plasma was not altered by increasing the amount of protein supplementation. No toxic effect of CP, SPP or LP was evident even when supplemented at high protein concentrations. It is concluded that seminal plasma protein, if added to ram spermatozoa after sorting and before freezing, can improve post-thaw sperm quality and consequently the efficiency of sorting. This effect is most likely related to protection of the spermatozoa during freeze-thawing.

Degradation studies on biodegradable nanocomposite based on polycaprolactone/polycarbonate (80:20%) polyhedral oligomeric silsesquioxane

The development of biocompatible polymers has greatly advanced the field of tissue engineering. Some tissues can be propagated on a nondegradable scaffold. Tissue such as cartilage, however, is a complex tissue in which the chondrocytes require their own synthesized extracellular matrix (ECM) to function. Suitable scaffolds for tissue engineering cartilage should provide mechanical strength and degrade at a similar rate to that of cell growth and ECM production. We have developed a biodegradable nanocomposite based on polycaprolactone and polycarbonate polyurethane (PCU) with an incorporated polyhedral oligomeric silsesquioxane (POSS) (POSS modified Poly(caprolactone/carbonate) urethane/urea). Previous work on POSS incorporated into PCU (POSS-PCU) has been shown to possess good mechanical strength, elasticity and resistance to degradation. This series of experiments involved exposing this polymer to a selection of accelerated degradative solutions for up to 8 weeks. The samples were analyzed by infra-red spectroscopy, scanning electron microscopy, X-ray microanalysis, contact angle analysis, and stress-strain mechanical analysis. Degradation of hard and soft segments of the nanocomposite was evident by infra-red spectroscopy in all conditioned samples. POSS nanocage degradation was evident in some oxidative/peroxidative systems accompanied by gross changes in surface topography and significant changes in mechanical properties. The hydrophobic polymer became more hydrophilic in all conditions. This biodegradable nanocomposite demonstrated steady degradation with protection of mechanical properties when exposed to hydrolytic enzymes and plasma protein fractions and exhibited more dramatic degradation by oxidation.This pattern may be potentially employed in tissue engineering scaffolds where controlled degradation and retained structural stability of the scaffold is required.

Identification of PDC-109-like protein(s) in buffalo seminal plasma

The FN-2 family of seminal plasma proteins represents the major protein fraction of bovine seminal plasma. These proteins also constitute the major seminal plasma proteins fraction in horse, goat and bison seminal plasma and are present in pig, rat, mouse, hamster and human seminal plasma. BSP-A1 and BSP-A2, the predominant proteins of the FN-2 family, are collectively termed as PDC-109. Fn-2 proteins play an important role in fertilization, including sperm capacitation and formation of oviductal sperm reservoirs. Significantly, BSP proteins were also shown to have negative effects in the context of sperm storage. No conclusive evidence for the presence of buffalo seminal plasma protein(s) similar to PDC-109 exists. Studies with buffalo seminal plasma indicated that isolation and identification of PDC-109-like protein(s) from buffalo seminal plasma by conventional methods might be difficult. Thus, antibodies raised against PDC-109 isolated, and purified from cattle seminal plasma, were used for investigating the presence of PDC-109-like protein(s) in buffalo seminal plasma. Buffalo seminal plasma proteins were resolved on SDS-PAGE, blotted to nitro cellulose membranes and probed for the presence of PDC-109-like protein(s) using the PDC-109 antisera raised in rabbits. A distinct immunoreactive band well below the 20-kDa regions indicated the presence of PDC-109-like protein(s) in buffalo seminal plasma.

Megalin contributes to the early injury of proximal tubule cells during nonselective proteinuria

Megalin, a member of the LDL receptor family, is expressed on the apical membrane of proximal tubules and serves as an endocytic scavenger of filtered proteins and hence might contribute to the tubule injury as a consequence of glomerular disease. To study its role, we crossed megalin knockout mosaic mice (lacking megalin expression in 60% of proximal tubule cells) with NEP25 mice (a transgenic line expressing human CD25 in the podocyte). Treatment of this transgenic mouse with the immunotoxin causes nephrotic syndrome, focal segmental glomerulosclerosis and tubule-interstitial injury. Following this treatment, the double transgenic mice had massive non-selective proteinuria and mild glomerular and tubular injury. Comparison of megalin-containing to megalin-deficient proximal tubule cells within each kidney showed that albumin, immunoglobulin light chain, IgA and IgG were preferentially accumulated in proximal tubule cells expressing megalin. Tubule injury markers such as heme-oxygenase-1, monocyte chemoattractant protein-1 and cellular apoptosis were also preferentially found in these megalin-expressing cells. These results show that megalin plays a pivotal role in the reabsorption of small to large molecular size proteins and provides direct in vivo evidence that reabsorption of filtered proteins triggers events leading to tubule injury.

Dietary plasma proteins, the intestinal immune system, and the barrier functions of the intestinal mucosa1

ABSTRACTThe intestinal mucosa contributes to homeostasis by preventing the entrance of biological and chemical agents across the epithelium that could alter the stability of the system. This protective function is especially important at the time of weaning, when animals are exposed to infectious agents and to numerous stresses such as the change of environment and diet. Diets supplemented with spray-dried plasma or plasma protein fractions have been shown to improve growth performance of farm animals and have been proposed as an alternative to antibiotics. In this review, we summarize our findings on the mechanism of action of dietary plasma proteins using a rat model of intestinal inflammation, based on the administration of Staphylococcus aureus enterotoxin B (SEB). Staphylococcal enterotoxin B activates the gut-associated lymphoid tissue (GALT), increasing T-lymphocytes in Peyer's patches and the number of activated T lymphocytes in mesenteric lymph nodes (organized GALT). In the lamina propria SEB increased cytotoxic T δγ and natural killer cell populations of the diffuse GALT. Staphyloccocal enterotoxin B significantly increased proinflammatory cytokines in Peyer's patches and mucosa. Plasma protein supplements modulated the mucosal immune response in organized and diffuse GALT, protecting GALT from possible excessive activation by the SEB challenge. These effects are accompanied by a reduction of proinflammatory cytokine production, supporting the view that changes in cytokine production mediate the effects of dietary plasma proteins during intestinal inflammation. The increase in mucosal permeability and intestinal secretion induced by SEB was associated with decreased expression of mucosal tight-junction and adherent-junction proteins. Both plasma and plasma protein fractions prevented the effects of SEB on intestinal permeability, thus reducing the exposure of the host to microbial and food antigens across the interstitial space. These findings indicate that dietary plasma proteins modulate functional and structural properties of the intestinal mucosa.

Dengue and Chikungunya viruses in blood donations: risks to the blood supply?

Dengue and Chikungunya viruses in blood donations: risks to the blood supply?

Proteomics as a tool for optimization of human plasma protein separation

The application of proteomics technology in purification of proteins from human plasma and for characterization of plasma-derived therapeutics has been recently discussed. However, until now, the impact of this technology on the plasma protein fractionation and analysis of the final product has not been realized. In the present work, we demonstrate the use of proteomic techniques the monitoring of the first step of the plasma fractionation by use of anion-exchange chromatography. This chromatographic method is frequently used in the purification scheme for isolation of vitamin K dependent clotting factors II, VII, IX and X, and clotting inhibitors protein C and protein S, as well as inter-alpha inhibitor proteins (IaIp). After the removal of immunoglobulin G and non-binding proteins in the flow-through fraction, albumin and weakly bound proteins were eluted with low concentration of sodium chloride. The proteins that strongly bind to the anion-exchange column were eluted by higher salt concentrations. The fractions of interest were analyzed, and proteins were identified by LC–ESI-MS/MS. By use of this method, not only candidates for therapeutic concentrates, but also some potentially harmful components were identified. This strategy was very helpful for further process optimization, fast identification of target proteins with relatively low abundance, and for the design of subsequent steps in their removal or purification.

Isoform Analysis of LC−MS/MS Data from Multidimensional Fractionation of the Serum Proteome

We developed a visualization approach for the identification of protein isoforms, precursor/mature protein combinations, and fragments from LC-MS/MS analysis of multidimensional fractionation of serum and plasma proteins. We also describe a pattern recognition algorithm to automatically detect and flag potentially heterogeneous species of proteins in proteomic experiments that involve extensive fractionation and result in a large number of identified serum or plasma proteins in an experiment. Examples are given of proteins with known isoforms that validate our approach and present a subset of precursor/mature protein pairs that were detected with this approach. Potential applications include identification of differentially expressed isoforms in disease states.

Albumin Is the Main Nucleophilic Target of Human Plasma: A Protective Role Against Pro-atherogenic Electrophilic Reactive Carbonyl Species?

The aim of this work was to study the metabolic fate of 4-hydroxy- trans-2-nonenal (HNE) in human plasma, which represents the main vascular site of reactive carbonyl species (RCS) formation and where the main pro-atherogenic target proteins are formed. When HNE was spiked in human plasma, it rapidly disappeared (within 40 s) and no phase I metabolites were detected, suggesting that the main fate of HNE is due to an adduction mechanism. HNE consumption was then monitored in two plasma fractions: low molecular weight plasma protein fractions (<10 kDa; LMWF) and high molecular weight plasma protein fractions (>10 kDa; HMWF). HNE was almost stable in LMWF, while in HMWF it was consumed by almost 70% within 5 min. Proteomics identified albumin (HSA) as the main protein target, as further confirmed by a significantly reduced HNE quenching of dealbuminated plasma. LC-ESI-MS/MS analysis identified Cys34 and Lys199 as the most reactive adduction sites of HSA, through the formation of a Michael and Schiff base adducts, respectively. The rate constant of HNE trapping by albumin was 50.61 +/- 1.89 M (-1) s (-1) and that of Cys34 (29.37 M (-1) s (-1)) was 1 order of magnitude higher with respect to that of GSH (3.81 +/- 0.17 M (-1) s (-1)), as explained by molecular modeling studies. In conclusion, we suggest that albumin, through nucleophilic residues, and in particular Cys34, can act as an endogenous detoxifying agent of circulating RCS.

Effect of Seminal Plasma Protein on Postthaw Viability and Fertility of Arctic Char Spermatozoa

AbstractSeminal plasma protein of Arctic char Salvelinus alpinus was characterized using sodium dodecyl sulfate (SDS) gel electrophoresis. Twelve protein bands with molecular weights of 7.2, 12.4, 15.3, 20.0, 20.4, 22.6, 39.4, 66.3, 74.0, 92.0, 94.5, and 130.1 kilodaltons (kDa) were detected. The effect of total seminal plasma protein and protein fractions of three categories (&lt;50, 50–100, and &gt;100 kDa) on postthaw sperm motility, viability, and fertility was tested. Incorporation of total seminal plasma protein, the fraction greater than 100 kDa, or the fraction less than 50 kDa into the semen extender (300 mmol of glucose/L of water, plus 10% methanol) had a deleterious effect on postthaw sperm motility, viability, and fertility in comparison with spermatozoa frozen in the semen extender only. However, adding the 50‐100‐kDa fraction of seminal plasma protein to the semen extender did not affect the postthaw sperm motility and fertility relative to spermatozoa frozen in the extender only. Further experiments are needed to test the effect of different concentrations of seminal plasma proteins alone or in a combination with other seminal plasma constituents on sperm physiology and viability during short‐term storage and cryopreservation.

Pathogen inactivation: making decisions about new technologies

Methods to remove and inactivate pathogens, used extensively in the manufacture of plasma protein fractions, have all but eliminated transmission of infectious agents by these products. 1 Technologies for reducing the risk of infection from single donor blood components have not been embraced as enthusiastically. Several methods have been introduced in Europe. Treatment with solvent/detergent (S/D) or methylene blue have both been applied to plasma components, and psoralen treatment of platelets (PLTs) has begun in several countries. 2-4 Although S/D-treated pooled plasma has been approved for use in the United States and Canada, none of these methods has been adopted for single-donor products in North America. Reasons for slow acceptance include 1) the current safety of the volunteer blood supply; 2) the success of surveillance and development of screening tests to deal with emerging pathogens; 3) the inability of current technologies to inactivate some agents such as spores, prions, and certain small nonencapsulated viruses; 4) concerns regarding remote risks from the residual chemical agents used during the pathogen inactivation (PI) process; 5) absence of any single method to treat whole blood or all components; and 6) the costeffectiveness of these technologies especially compared to strategies to reduce noninfectious risks of transfusion. 5

Typ-I-Allergie auf Seminalplasma

Human seminal plasma allergy (HSPA) is a rare allergic reaction to specific protein fractions of seminal plasma, whereof PSA seems to be a relevant allergen. Predominantly Type I-immunoreactions can occur. The main symptoms are localized and generalized urticaria and sometimes anaphylactic symptoms. The diagnosis is based on history, skin tests and on the determination of specific IgE-levels for (un)fractionated seminal plasma. Here we report a patient with recurrent episodes of generalized urticaria after unprotected sexual intercourse and positive prick-test-reaction on seminal plasma.

Purification of equine IgG using membrane based enhanced hybrid bioseparation technique: A potential method for manufacturing hyperimmune antibody

Hyperimmune equine IgG is widely used as antivenom and anti-rabies agents. This article discusses a membrane based enhanced hybrid bioseparation technique for efficient and scalable purification of equine immunoglobulin G (IgG) from horse serum. This technique is an improved version of a standard hybrid bioseparation technique developed within our group earlier for fractionation of human plasma proteins (Ghosh. 2004. J Membr Sci 237: 109-117). In the presence of a high antichaotropic salt concentration, equine IgG is selectively and reversibly captured within a stirred cell membrane module from horse serum, partly due to precipitation and microfiltration, and partly due to hydrophobic interaction based membrane adsorption, while the impurities are washed out from the device. The reversibly sequestered IgG is then released by lowering the salt concentration which favor both dissolution of the precipitated IgG and desorption of the membrane bound IgG. The enhanced hybrid bioseparation technique improves the IgG recovery from the membrane module by switching from a stirring to non-stirring mode during the IgG release phase. It also reduces membrane fouling by an appropriate pH switch. The effects of operating conditions on equine IgG capture were first systematically studied. The enhanced hybrid bioseparation technique was followed by an ultrafiltration step to remove ammonium sulfate and low molecular weight impurities. The equine IgG purity obtained under optimized conditions was 88% and its recovery was over 90%, both being significantly higher than corresponding values obtained using currently used purification techniques.

Ten-year experience with preimplantation genetic diagnosis (PGD) at the New York University School of Medicine Fertility Center

We describe our experience of over 300 cycles of preimplantation genetic diagnosis (PGD) and report clinical pregnancy rates (35%-67%) that support using this technology to screen for genetic disorders and chromosomal abnormalities. In clinical practice for over ten years, PGD offers couples the earliest form of genetic screening and may help improve ongoing pregnancy rates in poor-prognosis patients.

SDS-polyacrylamide gel electrophoresis of buffalo bulls seminal plasma proteins and their relation with semen freezability

The objective of this study was to evaluate the protein profiles of seminal plasma in buffalo bulls and to examine their correlation with semen characteristics. Semen of 10 buffalo bulls were collected by a bovine artificial vagina. Semen characteristics (motility, morphology, viability and concentration) were recorded. A part of the semen sample (1 ml) was diluted by tris-egg yolk-glycerol extender, packed in French straws and was frozen in liquid nitrogen. The straws were later thawed and semen characteristics were compared with those of the fresh semen. Seminal plasma was harvested by centrifugation; treated with cold ethanol and then, underwent SDS-polyacrylamide gel electrophoresis (PAGE). Twenty five protein bands were identified on the gel, of which those of <35.5 kDa were prominent (72% of the bands). Of these protein fractions, 24.5 kDa was significantly correlated with sperm progressive motility in fresh and viability in frozen-thawed semen while 45 kDa bands were correlated with abnormal morphology in frozen-thawed semen; 55 kDa protein fractions were correlated with sperm viability of fresh semen. Progressive motility, viability and abnormal sperm morphology of frozen-thawed semen were highly correlated with these parameters in the fresh semen. In conclusion, seminal plasma protein fractions in buffalo bulls are similar to those reported in other animal species and have some correlations with semen characteristics before and after freezing.

Vasoactive mediators and hypotensive transfusion reactions

Vasoactive mediators and hypotensive transfusion reactions