The neutralizing effect of heparin on blood-derived antimicrobial compounds: impact on antibacterial activity and inflammatory response.

Acting through a combination of direct and indirect pathogen clearance mechanisms, blood-derived antimicrobial compounds (AMCs) play a pivotal role in innate immunity, safeguarding the host against invading microorganisms. Besides their antimicrobial activity, some AMCs can neutralize endotoxins, preventing their interaction with immune cells and avoiding an excessive inflammatory response. In this study, we aimed to investigate the influence of unfractionated heparin, a polyanionic drug clinically used as anticoagulant, on the endotoxin-neutralizing and antibacterial activity of blood-derived AMCs. Serum samples from healthy donors were pre-incubated with increasing concentrations of heparin for different time periods and tested against pathogenic bacteria (Acinetobacter baumannii, Enterococcus faecium, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus) and endotoxins from E. coli, K. pneumoniae, and P. aeruginosa. Heparin dose-dependently decreased the activity of blood-derived AMCs. Consequently, pre-incubation with heparin led to increased activity of LPS and higher values of the pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6). Accordingly, higher concentrations of A. baumannii, E. coli, K. pneumoniae, and P. aeruginosa were observed as well. These findings underscore the neutralizing effect of unfractionated heparin on blood-derived AMCs in vitro and may lead to alternative affinity techniques for isolating and characterizing novel AMCs with the potential for clinical translation.

In vitro Removal of Protein-Bound Retention Solutes by Extracorporeal Blood Purification Procedures.

INTRODUCTION: When the kidneys or liver fail, toxic metabolites accumulate in the patient's blood, causing cardiovascular and neurotoxic complications and increased mortality. Conventional membrane-based extracorporeal blood purification procedures cannot remove these toxins efficiently. The aim of this in vitro study was to determine whether commercial hemoperfusion adsorbers are suitable for removing protein-bound retention solutes from human plasma and whole blood as well as to compare the removal to conventional hemodialysis. METHODS: For in vitro testing of the removal of protein-bound substances, whole blood and plasma were spiked with uremic retention solutes (homocysteine, hippuric acid, indoxyl sulfate, 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid) and the toxins of liver failure (bilirubin, cholic acid, tryptophan, phenol). Subsequently, the protein binding of each retention solute was determined. The adsorption characteristics of the hemoperfusion adsorbers, Jafron HA and Biosky MG, both approved for the adsorption of protein-bound uremic retention solutes and Cytosorb, an adsorber recommended for adsorption of cytokines, were tested by incubating them in spiked whole blood or plasma for 1 h. Subsequently, the adsorption characteristics of the adsorbers were tested in a dynamic system. For this purpose, a 6-h in vitro hemoperfusion treatment was compared with an equally long in vitro hemodialysis treatment. RESULTS: Hippuric acid, homocysteine, indoxyl sulfate, and tryptophan were most effectively removed by hemodialysis. Bilirubin and cholic acid were removed best by hemoperfusion with Cytosorb. A treatment with Jafron HA and Biosky MG showed similar results for the adsorption of the tested retention solutes and were best for removing phenol. 3-Carboxy-4-methyl-5-propyl-2-furanpropionic acid could not be removed with any treatment method. DISCUSSION/CONCLUSION: A combination of hemodialysis with hemoperfusion seems promising to improve the removal of some toxic metabolites in extracorporeal therapies. However, some very strongly protein-bound metabolites cannot be removed adequately with the adsorbers tested.

Heparin enables the reliable detection of endotoxin in human serum samples using the Limulus amebocyte lysate assay.

The determination of lipopolysaccharide (endotoxin) in serum or plasma samples using Limulus amebocyte lysate (LAL)-based assays is currently not sufficiently reliable in clinical diagnostics due to numerous interfering factors that strongly reduce the recovery of LPS in clinical samples. The specific plasma components responsible for the endotoxin neutralizing capacity of human blood remain to be identified. There are indications that certain endotoxin-neutralizing proteins or peptides, which are part of the host defense peptides/proteins of the innate immune system may be responsible for this effect. Based on our finding that several antimicrobial peptides can be neutralized by the polyanion heparin, we developed a heparin-containing diluent for serum and plasma samples, which enables reliable quantification of LPS measurement in clinical samples using the LAL assay. In a preclinical study involving 40 donors, this improved protocol yielded an over eightfold increase in LPS recovery in serum samples, as compared to the standard protocol. This modified protocol of sample pretreatment could make LPS measurement a valuable tool in medical diagnostics.

An in vitro study on factors affecting endotoxin neutralization in human plasma using the Limulus amebocyte lysate test.

Endotoxin neutralization, caused by plasma components, makes it difficult to detect endotoxins in human blood. In this study, we investigated which factors influence the recovery of endotoxins using limulus ameobocyte lysate (LAL)-based assays. The individual factors that were examined in more detail were lipoprotein content, type of blood anticoagulation, kinetics and serum levels of divalent cations. Furthermore, it was investigated whether there is a direct correlation between LAL activity and monocyte activation. We could show that polyanionic heparin increases endotoxin recovery in blood, while citrate anticoagulation promotes endotoxin neutralization. Furthermore, we could show that the endotoxin activity in human plasma and serum decreases strongly over time. Time-dependent endotoxin neutralization reaches its maximum after 4-6 h incubation. By means of filtration tests we could determine that endotoxins in the plasma bind to lipoproteins but do not influence their activity. Comparative measurements have shown that high LAL activity of endotoxins in plasma simultaneously possesses high monocyte activating properties in whole blood. For the maximum recovery of endotoxins in human blood the physiological calcium and magnesium concentrations are sufficient. In this study, it was shown that the endotoxin neutralizing plasma components have a molecular weight similar to ß2-microglobulin (11.7 kDa). For the exact identification of the endotoxin neutralizing plasma components, which caused a modulation of the immunostimulating endotoxin activity, further investigations have to be carried out in the future.

Impact of anion exchange adsorbents on regional citrate anticoagulation.

INTRODUCTION: Heparin and citrate are commonly used anticoagulants in membrane/adsorption based extracorporeal liver support systems. However, anion exchange resins employed for the removal of negatively charged target molecules including bilirubin may also deplete these anticoagulants due to their negative charge. The aim of this study was to evaluate the adsorption of citrate by anion exchange resins and the impact on extracorporeal Ca2+ concentrations. METHODS: Liver support treatments were simulated in vitro. Citrate and Ca2+ concentrations were measured pre and post albumin filter as well as pre and post adsorbents. In addition, batch experiments were performed to quantify citrate adsorption. RESULTS: Pre albumin filter target Ca2+ concentrations were reached well with only minor deviations. Citrate was adsorbed by anion exchange resins, resulting in a higher Ca2+ concentration downstream of the adsorbent cartridges during the first hour of treatment. CONCLUSIONS: The anion exchange resin depletes citrate, leading to an increased Ca2+ concentration in the extracorporeal circuit, which may cause an increased risk of clotting during the first hour of treatment. An increase of citrate infusion during the first hour of treatment should therefore be considered to compensate for the adsorption of citrate.

Ex vivo investigation on internal tunnel approach/internal resin infiltration and external nanosilver-modified resin infiltration of proximal caries exceeding into dentin.

This ex vivo proof-of-concept study aimed to investigate the effect of nanosilver particles (AgNP) added to a conventional infiltrant resin (Icon) on external penetration into natural proximal enamel caries exceeding into dentin after internal tunnel preparation and internal infiltration. Carious lesions (ICDAS codes 2/3) of extracted human (pre-)molars revealing proximal caries radiographically exceeding into dentin (E2/D1 lesions) were preselected. Then, 48 of those specimens showing demineralized areas transcending the enamel-dentin border as assessed by means of near-infrared light transillumination (DIAGNOcam) were deproteinized (NaOCl, 5%). Using an internal tunnel approach, occlusal cavities central to the marginal ridge were prepared. Excavation of carious dentin, total etch procedure (H3PO4, 40%), and internal resin infiltration (FITC-labeled) followed, along with final restorations (flowable composite resin). Outer lesion surfaces were etched (HCl, 15%) prior to external infiltration (RITC-labeled). Group 1 (control; n = 24) used non-modified infiltrant, while an infiltrant/AgNP mixture (20 nm; 5.5 wt%) was used with experimental Group 2 (n = 24). Non-infiltrated pores of cut lesions were stained (Berberine), and specimens were analyzed using confocal laser scanning microscopy. Compared to the non-filled infiltrant, incorporation of AgNP had no effect on the resin's external penetration. Between the groups, no significant differences regarding internal or external infiltration could be detected, and non-infiltrated lesion areas did not differ significantly (p>0.109; t-test). The internal tunnel preparation in combination with both an internal resin infiltration and an additional external infiltration approach using a nanosilver-modified infiltrant resin leads to increased infiltrated lesion areas, thus occluding and adhesively stabilizing the porous volume of the demineralized enamel. While exerting antimicrobial effects by the nanosilver particles, this approach should have the potential as a viable treatment alternative for proximal lesions extending into dentin, thus avoiding the sacrifice of sound enamel, postponing the frequently inevitable restoration/re-restoration cycle of conventional proximal caries treatment, and improving dental health.

Cytokine Removal in Extracorporeal Blood Purification: An in vitro Study.

BACKGROUND/AIMS: Cytokines are among the main target substances that have to be removed effectively in order to improve the patient's health status in the treatment of sepsis, septic shock, and liver diseases. Although there are various medical devices commercially available, the success of their clinical use is limited. The aim of this in vitro study was to compare 3 different medical devices with respect to their clearance for the cytokines interleukin-6 (IL-6), IL-8, IL-1ß, and tumor necrosis factor alpha. The medical devices that were tested are the whole blood adsorbent CytoSorb, the high cutoff filter EMiC2, and the hemofilter HemofeelCH 1.8. METHODS: The study was carried out on the multiFiltrate machine with 1 L human plasma for 8 h. Samples for cytokine quantification were taken at defined time points from the plasma pool. Each experiment was conducted in triplicates, and clearance was calculated for all tested cytokines. RESULTS: All 3 medical devices showed good cytokine removal. The highest clearance for all cytokines was achieved by hemoperfusion with Cytosorb. IL-8 and IL-6 clearance were higher with Hemofeel (continuous venovenous hemodiafiltration) than with EMiC2 (continuous venovenous hemodialysis) because the polymethyl methacrylate (PMMA)-based membrane Hemofeel is able to remove these 2 cytokines by adsorption. Protein and albumin loss was highest withCytosorb and lowest with EMiC2. CONCLUSION: The mechanisms of cytokine removal by blood purification include convection, diffusion, and adsorption. PMMA-based filters are able to combine all 3 mechanisms for certain cytokines. Cytosorb showed the best adsorption kinetics, while dialysis with polystyrene-based membranes offers the best biocompatibility because they do not show any unspecific adsorption of other plasma components.

Blood Compatibility-An Important but Often Forgotten Aspect of the Characterization of Antimicrobial Peptides for Clinical Application.

Acylation of antimicrobial peptides mimics the structure of the natural lipopeptide polymyxin B, and increases antimicrobial and endotoxin-neutralizing activities. In this study, the antimicrobial properties of lactoferrin-based LF11 peptides as well as blood compatibility as a function of acyl chain length were investigated. Beyond the classical hemolysis test, the biocompatibility was determined with human leukocytes and platelets, and the influence of antimicrobial peptides (AMPs) on the plasmatic coagulation and the complement system was investigated. The results of this study show that the acylation of cationic peptides significantly reduces blood tolerance. With increasing acyl chain length, the cytotoxicity of LF11 peptides to human blood cells also increased. This study also shows that acylated cationic antimicrobial peptides are inactivated by the presence of heparin. In addition, it could be shown that the immobilization of LF11 peptides leads to a loss of their antimicrobial properties.

Influence of citrate concentration on the activation of blood cells in an in vitro dialysis setup.

BACKGROUND: Regional citrate anticoagulation has been associated with enhanced biocompatibility in hemodialysis, but the optimal dose of citrate remains to be established. Here, we compared parameters related to cellular activation during in vitro dialysis, using two doses of citrate. METHODS: Human whole blood, anticoagulated with either 3 mM or 4 mM of citrate, was recirculated in an in vitro miniaturized dialysis setup. Complement (C3a-desArg), soluble platelet factor 4 (PF4), thromboxane B2 (TXB2), myeloperoxidase (MPO), as well as platelet- and red blood cell-derived extracellular vesicles (EV) were quantified during recirculation. Dialyzer fibers were examined by scanning electron microscopy after recirculation to assess the activation of clotting and the deposition of blood cells. RESULTS: Increases in markers of platelet and leukocyte activation, PF4, TXB2, and MPO were comparable between both citrate groups. Complement activation tended to be lower at higher citrate concentration, but the difference between the two citrate groups did not reach significance. A strong increase in EVs, particularly platelet-derived EVs, was observed during in vitro dialysis for both citrate groups, which was significantly less pronounced in the high citrate group at the end of the experiment. Assessment of dialyzer clotting scores after analysis of individual fibers by scanning electron microscopy revealed significantly lower scores in the high citrate group. CONCLUSIONS: Our data indicate that an increase in the citrate concentration from 3 mM to 4 mM further dampens cellular activation, thereby improving biocompatibility. A concentration of 4 mM citrate might therefore be optimal for use in clinical practice.

Removal of stabilizers from human serum albumin by adsorbents and dialysis used in blood purification.

INTRODUCTION: Human serum albumin (HSA) is a monomeric multi-domain protein that possesses an extraordinary binding capacity. It plays an important role in storing and transporting endogenous substances, metabolites, and drugs throughout the human circulatory system. Clinically, HSA is used to treat a variety of diseases such as hypovolemia, shock, burns, hemorrhage, and trauma in critically ill patients. Pharmaceutical-grade HSA contains the stabilizers sodium caprylate and N-acetyltryptophanate to protect the protein from oxidative stress and to stabilize it for heat treatment which is applied for virus inactivation. MATERIAL AND METHODS: The aim of this study was to determine if the two stabilizers can be depleted by adsorbent techniques. Several, adsorbents, some of them are in clinical use, were tested in batch and in a dynamic setup for their ability to remove the stabilizers. Furthermore, the removal of the stabilizers was tested using a pediatric high flux dialyzer. RESULTS: The outcome of this study shows that activated charcoal based adsorbents are more effective in removal of N-acetylthryptophanate, whereas polystyrene based adsorbents are better for the removal of caprylate from HSA solutions. An adsorbent cartridge which contains a mix of activated charcoal and polystyrene based material could be used to remove both stabilizers effectively. After 4 hours treatment with a high flux dialyzer, N-acetyltryptophanate was totally removed whereas 20% of caprylate remained in the HSA solution.

Clearance of Selected Plasma Cytokines with Continuous Veno-Venous Hemodialysis Using Ultraflux EMiC2 versus Ultraflux AV1000S.

BACKGROUND: High cutoff hemofilters might support the restoration of immune homeostasis in systemic inflammation by depleting inflammatory mediators from the circulation. METHODS: Interleukin (IL)-6, IL-8, IL-10, and tumor necrosis factor-alpha depletion was assessed in 30 sepsis patients with acute renal failure using continuous veno-venous hemodialysis with high cutoff versus standard filters (CVVHD-HCO vs. CVVHD-STD) over 48 h. RESULTS: The transfer of IL-6 and IL-8 was significantly higher for CVVHD-HCO, as shown by increased IL-6 and IL-8 effluent concentrations. The mean plasma cytokine concentrations decreased over time for all cytokines without detectable differences for the treatment modalities. No transfer of albumin was observed for either of the filters. C-reactive protein remained stable over time and did not differ between CVVHD-HCO and CVVHD-STD, while procalcitonin decreased significantly over 48 h for both treatment modalities. CONCLUSION: CVVHD-HCO achieved enhanced removal of IL-6 and IL-8 as compared to CVVHD-STD, without differentially reducing plasma cytokine levels.

Removal of bile acids by extracorporeal therapies: an in vitro study.

INTRODUCTION: Bile acids (BAs) accumulating in the circulation in patients with liver failure are considered to be responsible for pruritus, which strongly impairs quality of life of the affected patients. The aim of this study was the in vitro characterization of different BAs regarding their removability with high-flux dialysis as well as with different adsorbents, and the evaluation of their binding to plasma proteins. METHODS: Dialysis experiments were conducted in pediatric circuits with human plasma. For the adsorption studies, batch tests using 10% adsorbent in spiked human plasma were carried out. The binding of BAs to plasma compounds was determined by centrifugation of spiked plasma through spin columns. Sieving coefficients were determined using an albumin filter and a high-flux dialyzer. RESULTS: With high-flux dialysis, only hydrophilic BAs such as glycocholic and taurocholic acid could be removed efficiently, while all tested BAs were removed by adsorption. CONCLUSIONS: In conclusion, the hydrophilicity of BAs plays a major role in their removability using extracorporeal approaches. Adsorption-based systems offer particular advantages for the removal of hydrophobic BAs.

The role of ionized calcium and magnesium in regional citrate anticoagulation and its impact on inflammatory parameters.

INTRODUCTION: Regional anticoagulation with citrate has been found to be superior to heparin in terms of biocompatibility, and numerous protocols for regional citrate anticoagulation have been published, while a consensus on the target concentration of ionized calcium (Ca2+) in the extracorporeal circuit has not been reached so far. METHODS: The aim of this in vitro study was to assess the impact of different citrate concentrations on coagulation as well as on complement activation and cytokine secretion and to investigate the impact of ionized magnesium (Mg2+) on these parameters. RESULTS: We found that citrate effectively reduced coagulation, complement activation, and cytokine secretion in a dose-dependent manner and that a target Ca2+ concentration of 0.2-0.25 mM was required for efficient anticoagulation. Mg2+ triggered complement activation as well as interleukin (IL)-1ß secretion in lipopolysaccharide (LPS)-stimulated whole blood in a dose-dependent manner and independently of Ca2+. Additionally, it was found to reduce activated clotting time (ACT) in samples with low Ca2+ levels, but not at physiological Ca2+. CONCLUSIONS: Taken together, our data support the notion that regional citrate anticoagulation results in decreased release of inflammatory mediators in the extracorporeal circuit, requiring the depletion of both, Ca2+ and Mg2+.

A new integrated technique for the supportive treatment of sepsis.

Although there has been continuous, intensive research for many years in the field of sepsis treatment, currently available treatment options are limited, and there is still a lack of systems that efficiently remove endotoxins as well as mediators. Here, we discuss a newly developed, integrated technique that combines different aspects for their use in extracorporeal blood purification for the supportive treatment of liver failure and sepsis.

Low-dose polymyxin: an option for therapy of Gram-negative sepsis.

Endotoxins are the major components of the outer membrane of most Gram-negative bacteria and are one of the main targets in inflammatory diseases. The presence of endotoxins in blood can provoke septic shock in case of pronounced immune response. Here we show in vitro inactivation of endotoxins by polymyxin B (PMB). The inflammatory activity of the LPS-PMB complex in blood was examined in vitro in freshly drawn blood samples. Plasma protein binding of PMB was determined by ultracentrifugation using membranes with different molecular cut-offs, and PMB clearance during dialysis was calculated after in vitro experiments using the AV1000S filter. The formed LPS-PMB complex has lower inflammatory activity in blood, which results in highly reduced cytokine secretion. According to in vitro measurements, the appropriate plasma level of PMB for LPS inactivation is between 100 and 200 ng/ml. Furthermore, the combination of cytokine removal by adsorbent treatment with LPS inactivation by PMB dosage leads to strong suppression of inflammatory effects in blood in an in vitro model. Inactivation of endotoxins by low-dose intravenous PMB infusion or infusion into the extracorporeal circuit during blood purification can be applied to overcome the urgent need for endotoxin elimination not only in treatment of sepsis, but also in liver failure.

Adsorption of Selected Antibiotics to Resins in Extracorporeal Blood Purification.

BACKGROUND/AIMS: Extracorporeal blood purification systems (EBS) use specific adsorbents for the elimination of toxins and cytokines. The aim of this study was to test different adsorbents for their ability to reduce antibiotics in parallel to extracorporeal blood purification therapy. METHODS: The in vitro adsorption experiments were carried out in human plasma with a newly established hydrophobic resin (Amberchrom CG161c) and adsorbents commercially available and approved in the clinics. The concentration of antibiotic was chosen equivalent to the recommended therapeutic dosage applied intravenously and was measured in plasma using ELISA test kits and high-performance liquid chromatography methods. RESULTS: The adsorbent that reduced all tested antibiotics in plasma close to the detection limit was the dia MARS AC250, which is an activated charcoal involved in the Molecular Adsorbents Recirculation System. CONCLUSION: For better antibiotic monitoring in sepsis treatment, further investigations have to be performed to determine the clearance rate of antibiotics by different EBS devices.

Characterization of Adsorbents for Cytokine Removal from Blood in an In Vitro Model.

INTRODUCTION: Cytokines are basic targets that have to be removed effectively in order to improve the patient's health status in treating severe inflammation, sepsis, and septic shock. Although there are different adsorbents commercially available, the success of their clinical use is limited. Here, we tested different adsorbents for their effective removal of cytokines from plasma and the resulting effect on endothelial cell activation. METHODS: The three polystyrene divinylbenzene (PS-DVB) based adsorbents Amberchrom CG161c and CG300m and a clinically approved haemoperfusion adsorbent (HAC) were studied with regard to cytokine removal in human blood. To induce cytokine release from leucocytes, human blood cells were stimulated with 1 ng/ml LPS for 4 hours. Plasma was separated and adsorption experiments in a dynamic model were performed. The effect of cytokine removal on endothelial cell activation was evaluated using a HUVEC-based cell culture model. The beneficial outcome was assessed by measuring ICAM-1, E-selectin, and secreted cytokines IL-8 and IL-6. Additionally the threshold concentration for HUVEC activation by TNF-α and IL-1ß was determined using this cell culture model. RESULTS: CG161c showed promising results in removing the investigated cytokines. Due to its pore size the adsorbent efficiently removed the key factor TNF-α, outperforming the commercially available adsorbents. The CG161c treatment reduced cytokine secretion and expression of cell adhesion molecules by HUVEC which underlines the importance of effective removal of TNF-α in inflammatory diseases. CONCLUSION: These results confirm the hypothesis that cytokine removal from the blood should approach physiological levels in order to reduce endothelial cell activation.

Pore size--a key property for selective toxin removal in blood purification.

PURPOSE: Extracorporeal blood purification systems based on combined membrane/adsorption technologies are used in acute liver failure to replace detoxification as well as to remove inflammatory mediators in sepsis patients. In addition to coating and chemical modification of the surface, pore size significantly controls the selectivity of adsorption materials. METHODS: This study addresses the adsorption of albumin bound liver toxins, cytokines, and representative plasma compounds on three adsorbents which differ only in pore size distribution. All three adsorbents are based on hydrophobic poly(styrene-divinylbenzene) copolymer matrices and have mean pore sizes of 15, 30, and 100 nm. RESULTS: The pores of adsorbents act as molecular sieves and prevent the entry of molecules that are larger than their molecular cut-off. The results of this study reveal that adsorbents based on styrene-divinylbenzene polymers with 15 nm pores are suitable for cytokine removal, and the same adsorbents with 30-40 nm pores are the best choice for the removal of albumin-bound toxins in the case of liver failure. Adsorbents with very large pores lack selectivity which leads to uncontrolled adsorption of all plasma proteins. Therefore, hydrophobic adsorbents with large pores offer inadequate plasma compatibility and do not fulfill the requirements for blood purification. CONCLUSIONS: Biocompatibility and efficiency of adsorbents used for blood purification can improved by fine tuning of adsorbent surface pore distributions.

Adsorption of the inflammatory mediator high-mobility group box 1 by polymers with different charge and porosity.

High-mobility group box 1 protein (HMGB1) is a conserved protein with a variety of biological functions inside as well as outside the cell. When released by activated immune cells, it acts as a proinflammatory cytokine. Its delayed release has sparked the interest in HMGB1 as a potential therapeutic target. Here, we studied the adsorption of HMGB1 to anionic methacrylate-based polymers as well as to neutral polystyrene-divinylbenzene copolymers. Both groups of adsorbents exhibited efficient binding of recombinant HMGB1 and of HMGB1 derived from lipopolysaccharide-stimulated peripheral blood mononuclear cells. The adsorption characteristics depended on particle size, porosity, accessibility of the pores, and charge of the polymers. In addition to these physicochemical parameters of the adsorbents, modifications of the molecule itself (e.g., acetylation, phosphorylation, and oxidation), interaction with other plasma proteins or anticoagulants (e.g., heparin), or association with extracellular microvesicles may influence the binding of HMGB1 to adsorbents and lead to preferential depletion of HMGB1 subsets with different biological activity.