Effects of poly(ethyleneglycol)-modified hemoglobin vesicles on N-formyl-methionyl-leucyl-phenylalanine-induced responses of polymorphonuclear neutrophils in vitro.

[Poly(ethyleneglycol)]-modified hemoglobin vesicles (PEG-HbV), a type of encapsulated hemoglobin, have been developed as artificial oxygen carriers and it is important to evaluate their blood compatibility. We studied the effects of PEG-HbV on human polymor phonuclear neutrophils (PMNs) in vitro, focusing on the functional responses to N-formyl-methionyl-leucyl-phenylalanine (fMLP) as an agonist. The pretreatment of the PMNs with PEG-HbV up to a concentration of 60 mg/dl Hb did not affect the fMLP-triggered chemotactic activity. In parallel to these results, the fMLP-induced upregulation of CD11b (Mac-1) levels on the PEG-HbV-pretreated PMNs was comparable to that of untreated cells. Furthermore, the pretreatment of the PMNs with the PEG-HbV even at 600 mg/dl Hb did not affect the gelatinase B (Matrix methalloproteinase-9 (MMP-9)) release, suggesting that the fMLP-induced release of secondary and tertiary granules was normal. In addition, the fMLP-triggered superoxide production of the PMNs was unchanged by the pretreatment with the PEG-HbV at 600 mg/dl Hb. Thus, these results suggest that PEG-HbV, at the concentrations studied, have no aberrant effects on the fMLP-triggered functions of human PMNs.     

Inhibition of in vitro platelet aggregation and release and fibrinolysis

Inhibition of in vitro platelet aggregation and release of contents of platelet granules is necessary in order to assess accurately platelet activation in vivo. This can be accomplished by using a variety of inhibitors added to blood collection containers. An additive mixture of citrate, theophylline, adenosine, and dipyridamole (CTAD) provides a practical alternative to a mixture of acid citrate dextrose (ACD), acetylsalicylic acid (aspirin), and prostaglandin E1 (PGE1) because of the stability problems associated with PGE1. Inhibition of in vitro fibrinolysis is essential for the accurate measurement of fibrin degradation products (FDP). This can be accomplished by using a mixture of thrombin, soybean trypsin, or aprotinin into which blood is collected. However, in patients receiving heparin, the fibrinolysis inhibitor mixture is ineffective unless it is supplemented with reptilase. With increasing use of recombinant tissue-type plasminogen activator therapy (rt-PA), an inhibitor such as D-phenylalanine-proline-arginine-chloromethylketone (PPACK) used as a blood collection additive is superior to a conventional protease inhibitor, such as aprotinin.     

Characterization of poly(ethylene glycol)-modified bovine hemoglobin by capillary zone electrophoresis

In this study capillary zone electrophoresis (CZE) was used to analyze the poly(ethylene glycol)-modified bovine hemoglobin(PEG-bHb). The results show that CZE separated the subunit of bovine hemoglobin based on the number of PEG conjugating to the protein surface, which makes it possible to evaluate the degree of modification of hemoglobin subunit; meanwhile, it also reflected the stability of PEG attaching to hemoglobin after incubating with hydroxylamine, which makes it successful to detect the distribution of attachment site and evaluate the stability of PEG on the surface of hemoglobin. As a simple, fast and accurate method, CZE is suitable to monitor the production procedures and quality control of the final products of the PEG-bHb.     

Effects of hemoglobin vesicles on resting and agonist-stimulated human platelets in vitro

Hemoglobin vesicles (HbV) are artificial oxygen carriers that encapsulate a concentrated hemoglobin (Hb) solution with a phospholipid bilayer membrane. The oxygen transporting ability of HbV in vivo has been demonstrated by the transfusion of HbV into hemorrhagic shock rodent models. However, the compatibility of HbV with human blood cells must be evaluated. Preincubation of platelets with concentrations of 20% or 40% HbV had no effect on the binding of PAC-1, a monoclonal antibody that detects activation-dependent conformational changes in alphaIIbbeta3 on platelets, or the surface expression of CD62P in whole blood. ADP-induced increases in PAC-1 binding were significantly enhanced by exposing the platelets to concentrations of either 20% or 40% HbV, whereas the ADP-induced increases in CD62P expression were not affected by HbV treatment at either concentration. Preincubation of platelet-rich plasma (PRP) with HbV minimally reduced the spontaneous release of TXB2 and RANTES, but did not significantly affect the formation of TXB2 or the release of RANTES and beta-TG in platelets stimulated with ADP. Similarly, preincubation of PRP with HbV minimally reduced the spontaneous release of RANTES but did not significantly affect the formation of TXB2 or the release of RANTES and beta-TG in platelets stimulated with collagen, although collagen-induced serotonin release tended to decrease with HbV pretreatment. These data suggest that the exposure of human platelets to high concentrations of HbV (up to 40%) in vitro did not cause platelet activation and did not adversely affect the formation and secretion of prothrombotic substances or proinflammatory substances triggered by platelet agonists, although one of the earliest events in ADP-induced platelet activation was slightly potentiated by HbV pretreatment at the doses tested. Taken together, these results imply that HbV, at concentrations of up to 40%, do not have any aberrant interactions with either unstimulated or agonist-induced platelets.     

Cationic proteins of human granulocytes Effects on human platelet aggregation and serotonin release

Immune-aggregate and thrombin-mediated [³H]serotonin release from human platelets are shown to be enhanced when platelets are preincubated with the antibacterial chymotrypsin-like cationic protein isolated from human granulocytes. The enhancement is dose dependent and inhibited by heating of the cationic protein. Release with chymotrypsin-like cationic protein alone was not observed, although the protein was shown to micro-aggregate platelets irreversibly by an ADP-dependent reaction. Platelet macro-aggregation induced by immune-aggregate was also enhanced by chymotrypsin-like cationic protein whereas platelet macro-aggregation induced by thrombin was inhibited competitively. Platelet micro-aggregation induced by chymotrypsin-like cationic protein was inhibited when preincubated for more than 5 min with a 2-fold molar excess of-1-antitrypsin. Chymotrypsin-like cationic protein interaction with several platelet reactions suggests a close relationship between neutrophils and platelets in the inflammatory process.     

Effects of heparin on platelet aggregation and release and thromboxane A2 production.

Heparin, when added to citrated platelet-rich plasma (PRP), caused potentiation of platelet aggregation and the release reaction induced by the aggregating agents adenosine diphosphate (ADP), arachidonic acid, collagen, and epinephrine. At low concentrations (4.7 x 10(-5) M) arachidonic acid failed to cause aggregation of platelets in citrated PRP. However, in the presence of heparin, the same concentration of arachidonic acid caused aggregation. Examination of PRP for the presence of thromboxane A2 (TxA2) by use of a bioassay revealed that heparin also stimulated release of TxA2. This finding indicated that platelets released more TxA2 when they were challenged by low concentrations of arachidonic acid in the presence of heparin than in its absence. Platelets were labeled with 3H-arachidonic acid and 14C-serotonin, and attempts were made to determine whether heparin stimulated the platelet release reaction first with subsequent increased production of TxA2, or alternatively, whether heparin stimulated TxA2 production first with subsequent enhancement of the release reaction. In view of the demonstrated simultaneous release of 14C-serotonin and 3H-arachidonic acid metabolites, it appeared that either release of 14C and 3H occurs concurrently or, even if one of these events is dependent on the other, both events take place in rapid succession. Timed sequential studies revealed that in the presence of arachidonic acid, the addition of heparin hastened the apparently simultaneous release of both 14C and 3H.     

Synthesis and properties of novel block copolymers containing poly(lactic-glycolic acid) and poly(ethyleneglycol) segments

A synthetic process for obtaining high-molecular-weight block copolymers containing poly(lacticglycolic acid) and poly(ethylene glycol) segments has been established. This process involves the reaction of poly(ethylene glycols) with phosgene, followed by polycondensation of the resulting ,ω-bis (chloroformates) with poly(lactic-glycolic acid) oligomers. The copolymers have been characterized for their molecular weight, solubility properties, water absorption and preliminarily thermal behaviour. All evidence points to the conclusion that the process described is a general one, enabling biodegradable polymers to be obtained tailor-made according to specific requirements.     

Effects of interferon-gamma on mobilization and release of eosinophil-derived RANTES

Eosinophils synthesize and release a number of cytokines and chemokines, including RANTES, a potent chemoattractant particularly for memory T cells and eosinophils. Long-term (>12 h) incubation with interferon-gamma (IFN-gamma) has been shown to activate eosinophils and induce expression of membrane receptors. We hypothesized that IFN-gamma mobilizes intracellular RANTES in eosinophils in advance of mediator release. Highly purified peripheral blood eosinophils were obtained from asthmatics and stimulated with IFN-gamma at 500 U/ml for time course analysis up to 2h. By specific ELISA, RANTES was detected in supernatants (80+/-15 pg per 2x10(6) cells) following 120min of stimulation. Immunoreactive RANTES in resting cells (5x10(7) eosinophils) was detected in two intracellular compartments in studies of subcellular fractionation by density gradient centrifugation. After 10 min IFN-gamma stimulation, RANTES immunoreactivity was confined to crystalloid granules. RANTES was redistributed from secretory granules to light-membrane fractions after 60 min of IFN-gamma incubation. Our data suggest that rapid mobilization and release of RANTES occurs from stimulated eosinophils. These findings may have important implications for the role of IFN-gamma in activating human eosinophils, particularly in severe chronic asthma or viral exacerbation of asthmatic inflammation, where this cytokine may play a role.     

Preparation and cytotoxic activity of poly(ethylene glycol)-modified poly(amidoamine) dendrimers bearing adriamycin

We have developed poly(amidoamine) (PAMAM) dendrimers that have poly(ethylene glycol) (PEG) grafts at all dendrimer chain ends. To obtain PEG-modified dendrimers with sites for conjugation of anticancer drugs for this study, we prepared PAMAM G4 dendrimers that have a glutamic acid (Glu) residue at every chain end of dendrimer; PEG chains were attached to amino groups of Glu residues. We then combined the anticancer drug adriamycin to side chains of the Glu residues using an amide bond, [PEG-Glu(ADR)-G4], or hydrazone bond, [PEG-Glu(NHN-ADR)-G4]. For the dendrimers bearing adriamycin through amide linkage, adriamycin was released only slightly at pH 7.4 and 5.5. Although a negligible level of release occurred at pH 7.4 for dendrimers with adriamycin via hydrazone linkage, a remarkable extent of adriamycin release was induced at pH 5.5, which corresponds to the pH of late endosome. These adriamycin-bearing dendrimers showed much lower toxicity to HeLa cells than did free adriamycin. However, compared to PEG-Glu(ADR)-G4, PEG-Glu(NHN-ADR)-G4 exhibited 7 times higher cytotoxicity, suggesting the importance of pH-sensitive hydrazone linkage for high cytotoxicity. Furthermore, the PEG-modified dendrimers exhibited an equivalent level of toxicity to that of adriamycin-resistant SBC-3/ADR100 cells and their parent adriamycin-sensitive SBC-3 cells.     

Hemocompatibility and biofunctionality of two poly(2-(dimethylamino)ethyl methacrylate-co-poly(ethyleneglycol) copolymers

To mask the antigenic sites of cells for cell therapies, especially for blood transfusion, we investigated the hemocompatibility of two poly(2-(dimethylamino)ethyl methacrylate-co-poly(ethyleneglycol) compared with that of the homopolymer without PEG. Our strategy relies on the potential ability of these copolymers to self-assemble at the erythrocyte surface. The cationic sequence of the copolymer should be able to interact with the glycocalyx by ionic interaction. The other sequence, based on a polyethyleneglycol moiety, should prevent both nonspecific interactions and specific recognition of the biological surface. The hemocompatibility of these copolymers was assessed by analyzing alterations in human erythrocyte membrane viscoelasticity, morphology, granularity, and aggregation. Their properties to mask ABO system and three erythrocyte glycophorin sites were investigated. No alterations in the erythrocyte morphology were observed by confocal microscopy. On the other hand, a partial masking of different specific glycophorin sites leads to future optimization of the macromolecular structures of these functionalized copolymers.     

Effects of red wine and wine polyphenol resveratrol on platelet aggregation in vivo and in vitro.

Low to moderate consumption of red wine reportedly has a relatively greater benefit than other alcoholic beverages in the prevention of atherosclerosis and coronary heart disease (CHD). This beneficial effect is increasingly attributed to the polyphenol resveratrol, present in red wine. In the present study, we investigated the effects of resveratrol and red wine on aggregation of platelets isolated from healthy, normotensive male volunteers and in rabbits with experimental hypercholesterolemia. Platelet aggregation rate (PAR) was measured using Born's method. The results showed that aggregation of platelets from healthy subjects induced in vitro by collagen (5 microg/ml), thrombin (0.33 units/ml), and ADP (4 microM) was significantly inhibited by 10-1000 microM resveratrol, in a concentration-dependent manner. Hypercholesterolemic rabbits showed enhanced ADP-induced platelet aggregation; the average PAR increased from 39.5+/-5.9% in normal animals to 61.0+/-7.0% in the high-cholesterol fed group (n=8, p<0.001). Resveratrol (4 mg/kg/day) inhibited ADP-induced platelet aggregation in vivo by maintaining the PAR at 35.7+/-6.3% (vs. 39.5+/-5.9% for control rabbits, n=8, p=0.228), but had no effect on serum lipid levels. Similarly platelet aggregation in hypercholesterolemic rabbits was also inhibited when animals received intragastrically Chinese red wine (with or without alcohol, 4 ml/kg/day). These results suggest that resveratrol can inhibit platelet aggregation both in vitro and in vivo, which conceivably could be one of the mechanisms by which this red wine polyphenol exerts its cardioprotective effects.     

New pharmacological profiles of loratadine: Effects on platelet aggregation and histamine release from repository cells

Loratadine is endowed with antiallergic activity. It reduces both the immunological release of histamine in active anaphylaxis of isolated guinea-pig mast cells and in anti-IgE challenged human basophils as well as the non-immunological release of histamine induced by compound 48/80 and calcium ionophore A 23187 in isolated purified rat serosal mast cells. Loratadine was also found capable of directly reducing platelet aggregation induced by different agonists in a concentration-dependent fashion. It is capable of blocking the potentiation afforded by histamine on human platelet aggregation induced by thrombin, collagen and arachidonic acid. The present results suggest a broader profile for loratadine beyond its original role as a non-sedative anti-H₁-antihistaminic drug.     

Folate coupled poly(ethyleneglycol) conjugates of anionic poly(amidoamine) dendrimer for inflammatory tissue specific drug delivery

Folate receptor is overexpressed on the activated (but not quiescent) macrophages in both animal models and human patients with naturally occurring rheumatoid arthritis. The aim of this study was to prepare folate targeted poly(ethylene glycol) (PEG) conjugates of anionic dendrimer (G3.5 PAMAM) as targeted drug delivery systems to inflammation and to investigate its biodistribution pattern in arthritic rats. Folate-PEG-PAMAM conjugates, with different degrees of substitution were synthesized by a two-step reaction through a carbodiimide-mediated coupling reaction and loaded with indomethacin. Folate-PEG conjugation increased the drug loading efficiency by 10- to 20-fold and the in vitro release profile indicated controlled release of drug. The plasma pharmacokinetic parameters indicated an increased AUC, circulatory half-life and mean residence time for the folate-PEG conjugates. The tissue distribution studies revealed significantly lesser uptake by stomach for the folate-PEG conjugates, thereby limiting gastric-related side effect. The time-averaged relative drug exposure (r(e)) of the drug in paw for the folate-PEG conjugates ranged from 1.81 to 2.37. The overall drug targeting efficiency (T(e)) was highest for folate-PEG conjugate (3.44) when compared to native dendrimer (1.72). The folate-PEG-PAMAM conjugates are the ideal choice for targeted delivery of antiarthritic drugs to inflammation with reduced side-effects and higher targeting efficiency.     

Effects of the novel recombinant plasminogen activator BM 06.022 on human platelet aggregation in vitro

The present study was performed to evaluate effects of the novel recombinant plasminogen activator BM 06.022 on human platelet aggregation. BM 06.022 is an unglycosylated deletion variant of human tissue-type plasminogen activator. Aggregation was measured in platelet-rich plasma (PRP) from healthy human volunteers in response to adenosine diphosphate (ADP). Exposure of PRP to BM 06.022 induced a concentration-and time-dependent reduction of platelet aggregation compared with vehicle-control experiments. Incubation of high (3200 U/ml) concentrations of BM 06.022 for 0 to 15 min did not increase platelet aggregation. The platelet inhibitory effect of BM 06.022 could be reduced or abolished by aprotinin or chloromethylketone (PPACK), respectively, which is indicative of a plasmin-dependent mechanism. Concomitant incubation of BM 06.022, acetylsalicylic acid (ASA), and heparin decreased platelet aggregation less than BM 06.022 plus ASA alone, because heparin alone increased platelet aggregation. These findings demonstrate an inhibitory effect of BM 06.022 on platelet aggregation in citrated plasma, probably at least in part due to a plasmin-dependent mechanism.     

The curing reaction of poly(ether-sulfone)-modified epoxy resin

One of the useful methods to improve the toughness of epoxy resin is by mixing the resin with poly(ether-sulfone) (PES). In the present work, two hydroxyl-terminated PESs with molecular weights M _n = 28 600 and 4 200 were used for blending with epoxy resin. The curing reaction of diglycidyl ether/bisphenol-A (DGEBA) with 4,4′-diaminodiphenyl sulfone (DDS) in the presence of hydroxyl-terminated PES was studied by means of differential scanning calorimetry (DSC) and gel-permeation chromatography (GPC). For the DGEBA-DDS-PES system with a stoichiometric ratio of epoxy and amino groups the DSC experimental results showed that at a fixed molecular weight of PES the curing reaction rate decreases with increasing PES concentration. At a fixed PES concentration (in the range of between 0 and 20 wt.-% of PES), the DGEBA-DDS system modified with hydroxyl-terminated PES with lower molecular weight had a faster curing reaction rate at low conversion and a slower curing reaction rate at high conversion. The GPC results showed the evidence of etherification between low-molecular-weight PES with epoxy resin. However, very little etherification of high-molecular-weight hydroxyl-terminated PES with epoxy resin was found. Based on the experimental results, a curing reaction mechanism of DGEBA with DDS in the presence of hydroxyl-terminated PES is proposed.     

Hydrogels based on poly(ethylene oxide) and poly(tetramethylene oxide) or poly(dimethyl siloxane): synthesis, characterization, in vitro protein adsorption and platelet adhesion

In vitro protein adsorption, platelet adhesion and activation on new hydrogel surfaces, composed of poly(ethylene oxide) (PEO) and poly(tetramethylene oxide) (PTMO) or poly(dimethyl siloxane) (PDMS), were investigated. By varying PEO length (MW = 2000 or 3400), hydrophobic components (PTMO or PDMS) or polymer topology (block or graft copolymers), various physical hydrogels were produced. Their structures were verified by 1H NMR and ATR-IR and the molecular weights were determined by gel permeation chromatography. The hydrogels were soluble in a variety of organic solvents, while absorbed a significant amount of water with preserved three-dimensional structure by physical crosslinking. The dynamic contact angle measurement revealed that the surface hydrophilicity increased by incorporating longer PEO, PEO grafting, and adopting PDMS as a hydrophobic segment instead of PTMO. It was observed from in vitro protein adsorption study that the hydrogels exhibited significantly lower adsorption of human serum albumin (HSA), human fibrinogen (HFg), and IgG, when compared with Pellethane, a commercial polyurethane taken as a control. The hydrogels were attractive for HSA but not sensitive to HFg and IgG. And more than 65% of the proteins detected on the surfaces of the hydrogels were reversibly detached by being treated with an SDS solution. It was evident that the hydrogels synthesized in this study were much more resistant to platelet adhesion than the control, which might depend on the composition of proteins adsorbed on the surfaces and their degree of denaturation. Among the hydrogels tested, PEO3,4kPDMS exhibited albumin-rich and platelet-resistant surfaces, implying a potential candidate for biomaterial.