Red Blood Cell Binding Research Articles

Plasmodium falciparum: red blood cell binding studies using peptides derived from rhoptry-associated protein 2 (RAP2)

Plasmodium falciparum rhoptry-associated proteins 1 (RAP1) and 2 (RAP2) are antigens presenting themselves as candidates for a subunit malaria vaccine. RAP2 protein, non-overlapping, consecutive peptides were synthesised and tested in red blood cell (RBC) binding assays to identify their receptor–ligand interaction in recognising RAP2 regions involved in the in vitro merozoite invasion process. Four high activity binding peptides (HABPs) were identified in the resulting 20 peptides. Peptides 26220 ( 61NHFSSADE LIKYLEKTNINT 80), 26225 ( 161IKKN PFLRV LNKA STTTH AT 180) and 26229 ( 241RSVNNV ISKN KTLG LRKRSS 260) were located in the amino terminal and central part of the protein and HABP 26235 ( 361FLAEDFVELFDVTMDCYSRQ 380) was located at the carboxy terminal. All these HABPs showed saturable binding and presented dissociation constants between 500 and 950 nM; the number of binding sites per RBC ranged from 48 000 to 160 000. High binding peptides’ critical amino acids involved in RBC binding were determined by competition binding assays; their amino acids appear in bold in the sequences shown above. SDS-PAGE results showed that peptides 26220, 26225 and 26229 had at least two different sets of 62 and 42 kDa HABP receptors on RBCs and that peptide 26235 had at least two different sets of 77 and 62 kDa. HABPs inhibited in vitro merozoite invasion by between 54% and 94% at 200 μM, suggesting that these RAP2 peptides are involved in the in vitro P. falciparum invasion process.

In vitro pharmacokinetic study of the novel anticancer agent E7070: red blood cell and plasma protein binding in human blood.

E7070 is a novel sulfonamide anticancer agent that arrests the G(1)/S phase of the cell cycle. Preclinical and phase I studies have demonstrated non-linear pharmacokinetics (PK) of the drug. A population PK analysis revealed that the human plasma concentration-time data were best described by a three-compartment model with non-linear distribution. We have studied the in vitro interaction of 14C-radiolabeled E7070 with red blood cells (RBC) and its binding to plasma proteins in the concentration range where non-linearity in disposition was observed in humans to get more insight into the behavior of the drug. After the addition of E7070 to whole blood at 37 degrees C, the drug is taken up or binds to RBC in a concentration-dependent manner. The addition of sodium azide, however, did not result in a decrease of drug uptake by RBC, indicating passive diffusion processes. A non-linear increase in drug uptake was observed at incubation concentrations above 4 microg/ml E7070 in whole blood. This non-linearity was confirmed by lower partition coefficients between RBC and plasma at higher incubation concentrations (from 2.37 at 4 microg/ml to 0.31 at 200 microg/ml). The plasma protein binding of E7070 was high (98-99%) and linear in the concentration range studied (20-200 microg/ml). In conclusion, E7070 in whole blood is preferentially bound to RBC and exhibits high plasma protein binding. The non-linear distribution of E7070 in humans can be caused, in part at least, by saturable binding of E7070 to RBC.

Triggering of Human Parainfluenza Virus 3 Fusion Protein (F) by the Hemagglutinin-Neuraminidase (HN) Protein: an HN Mutation Diminishes the Rate of F Activation and Fusion

For human parainfluenza virus type 3 and many other paramyxoviruses, membrane fusion mediated by the fusion protein (F) has a stringent requirement for the presence of the homotypic hemagglutinin-neuraminidase protein (HN). With the goal of gaining further insight into the role of HN in the fusion process, we developed a simple method for quantitative comparison of the ability of wild-type and variant HNs to activate F. In this method, HN/F-coexpressing cells with red blood cells (RBC) bound to them at 4 degrees C are transferred to 22 degrees C, and at different times after transfer 4-guanidino-neu5Ac2en (4-GU-DANA) is added; this inhibitor of the HN-receptor interaction then releases all reversibly bound RBC but not those in which F insertion in the target membrane or fusion has occurred. Thus, the amount of irreversibly bound (nonreleased) RBC provides a measure of F activation, and the use of fluorescently labeled RBC permits microscopic assessment of the extent to which F insertion has progressed to fusion. We studied two neuraminidase-deficient HN variants, C28a, which has two mutations, P111S and D216N, and C28, which possesses the D216N mutation only. C28a but not C28 exhibits a slow fusion phenotype, although determination of the HNs' receptor-binding avidity (with our sensitive method, employing RBC with different degrees of receptor depletion) showed that the receptor-binding avidity of C28a or C28 HN was not lower than that of the wild type. The F activation assay, however, revealed fusion-triggering defects in C28a HN. After 10 and also 20 min at 22 degrees C, irreversible RBC binding was significantly less for cells coexpressing wild-type F with C28a HN than for cells coexpressing wild-type F with wild-type HN. In addition, F insertion progressed to fusion more slowly in the case of C28a HN-expressing cells than of wild-type HN-expressing cells. Identical defects were found for P111S HN, whereas for C28 HN, representing the 216 mutation of C28a, F activation and fusion were as rapid as for wild-type HN. The diminished fusion promotion capacity of C28a HN is therefore attributable to P111S, a mutation in the stalk region of the molecule that causes no decrease in receptor-binding avidity. C28a HN is the first parainfluenza virus variant found so far to be specifically defective in HN's F-triggering and fusion promotion functions and may contribute to our understanding of transmission of the activating signal from HN to F.

Newcastle disease virus HN protein alters the conformation of the F protein at cell surfaces.

Conformational changes in the Newcastle disease virus (NDV) fusion (F) protein during activation of fusion and the role of HN protein in these changes were characterized with a polyclonal antibody. This antibody was raised against a peptide with the sequence of the amino-terminal half of the F protein HR1 domain. This antibody immunoprecipitated both F(0) and F(1) forms of the fusion protein from infected and transfected cell extracts solubilized with detergent, and precipitation was unaffected by expression of the HN protein. In marked contrast, this antibody detected significant conformational differences in the F protein at cell surfaces, differences that depended upon HN protein expression. The antibody minimally detected the F protein, either cleaved or uncleaved, in the absence of HN protein expression. However, when coexpressed with HN protein, an uncleaved mutant F protein bound the anti-HR1 antibody, and this binding depended upon the coexpression of specifically the NDV HN protein. When the cleaved wild-type F protein was coexpressed with HN protein, the F protein bound anti-HR1 antibody poorly although significantly more than F protein expressed alone. Anti-HR1 antibody inhibited the fusion of R18 (octadecyl rhodamine B chloride)-labeled red blood cells to syncytia expressing HN and wild-type F proteins. This inhibition showed that fusion-competent F proteins present on surfaces of syncytia were capable of binding anti-HR1. Furthermore, only antibody which was added prior to red blood cell binding could inhibit fusion. These results suggest that the conformation of uncleaved cell surface F protein is affected by HN protein expression. Furthermore, the cleaved F protein, when coexpressed with HN protein and in a prefusion conformation, can bind anti-HR1 antibody, and the anti-HR1-accessible conformation exists prior to HN protein attachment to receptors on red blood cells.

Protection against experimental malaria associated with AMA-1 peptide analogue structures

One Plasmodium falciparum malaria antigen is an integral membrane protein called apical membrane antigen-1. High activity binding peptides to human red blood cells have been identified in this protein. 4337 is a conserved, non-immunogenic peptide with high activity red blood cell binding and its critical residues have already been identified. Peptide analogues (with amino acids having the same mass but different charge) were generated to change their immunogenic and protective characteristics. Three analogues having positive or negative immunological results were studied by nuclear magnetic resonance. The studied peptides all had an α-helix fragment, but in different peptide regions and extensions, except for randomly structured 4337. We show that altering a few amino acids induced immunogenicity and protectivity against experimental malaria and changed their three-dimensional structure, suggesting a better fit with immune system molecules and that modified peptides having better immunological properties can be included in the design of new malaria multi-component subunit-based vaccine.

Plasmodium vivax MSP-1 peptides have high specific binding activity to human reticulocytes

Plasmodium vivax merozoites have high preferential ability to interact with and invade reticulocytes, although these cells correspond to only 2% of the red blood cells (RBC) population. P. vivax merozoite surface protein-1 ( Pv-MSP-1) is believed to have an important role in attachment and invasion process. Using 88 non-overlapping 20-mer peptides, covering the entire Pv-MSP-1 Belem strain sequence, RBC and reticulocyte binding assays were performed. Fourteen sequences were identified with high specific binding activity to reticulocytes, but only three had high specific binding activity to mature erythrocytes. These peptides showed affinity constant values between 20 and 150 nM, indicating a strong interaction between these sequences and reticulocyte receptors. Critical residues in binding to reticulocytes for these peptides were determined by competition binding assays with glycine scanning analogues. All high binding peptides bind to reticulocyte surface proteins having a molecular mass of around 18–20 kDa which are not present in mature RBC. Interestingly, some high activity binding peptides (HABPs) are located close to the hypothesised 42 and 19 kDa fragment cleavage sites for this protein, suggesting that these sequences have an important role in target cell attachment and invasion process by Pv-MSP-1. HABPs may be clustered in two regions, with region I being located between amino acids 280–719, and region II between amino acids 1060–1599 with higher than 25% identity level. A P. falciparum MSP-1 antigenic domain binds to RBCs and inhibits parasite invasion. Peptides 1721 and 1724 bind with high activity to reticulocytes in homologous Pv-MSP-1, suggesting similar functions for these two sequences.

Sequence diversity and linkage disequilibrium within the merozoite surface protein-1 (Msp-1) locus of Plasmodium falciparum: a longitudinal study in Brazil.

The merozoite surface protein-1 (MSP-1) is a major vaccine candidate for the asexual blood stage of malaria. We examined both the extent of sequence diversity in block 17, the 3' end of Msp-1 gene coding for a 19-kDa polypeptide (MSP-1(19)) putatively involved in red blood cell binding, and the patterns of linkage disequilibrium between polymorphic sites throughout the Msp-1 locus. The parasite population sample consisted of Plasmodium falciparum isolates collected between 1985 and 1998 in Rondĵnia, an area of hypoendemic malaria transmission in the southwestern Brazilian Amazon. Results were summarized as follows. (1) Seven block-17 sequence variants or haplotypes were found among 130 isolates, including two new haplotypes (novel combinations of previously reported amino acid replacements), here named Brazil-1 (E-TSR-F) and Brazil-2 (Q-TSR-F). (2) As previously shown for other Msp-1 polymorphisms, frequencies of block-17 haplotypes displayed significant temporal variation. (3) Extensive linkage disequilibrium was demonstrated between neighboring dimorphic sites within block 17, as well as between polymorphisms at the 5' and 3' ends of Msp-1 (map distance range: 3.83-4.99 kb). (4) The overall patterns of linkage disequilibrium within Msp-1 remained stable over a period of nearly one decade, and examples of possible 'epidemic' expansion of parasites carrying particular Msp-1 alleles were found in the 1980s and 1990s. These results are discussed in relation to the population biology of P. falciparum and the development of malaria vaccines based on MSP-1.

Serine repeat antigen peptides which bind specifically to red blood cells

It has been reported that serine repeat antigen (SERA) binds directly to human erythrocyte membranes, inside-out vesicles and intact mouse erythrocytes. Similarly, mAbs specific against SERA are effective in blocking red blood cell (RBC) invasion by P. falciparum merozoites. Furthermore, the N-terminal recombinant SERA fragment inhibits the merozoite invasion of erythrocyte. In this study of 49 non-overlapping 20-residue-long peptides encompassing the whole SERA protein FCR3 strain, seven peptides having high RBC binding activity were found. Six of these peptides (three from the SERA N-terminal domain) are located in conserved regions and show affinity constants between 150 and 1100 nM, Hill coefficients between 1.5 and 3.0 and 30 000–120 000 binding sites per cell. Some of these peptides inhibited in vitro merozoite invasion of erythrocyte and intra-erythrocytic development. Residues which are critical in the binding to erythrocytes (in bold face), i.e. 6725 (YLK ETNNA ISF ES NSGSLEK K), 6733 (YALGS DIPEKCDT LAS NCFLS), 6737 (YD NI LV KM FK TNENNDKSEL I), 6746 (D QG NC DTSW IFASK YHLETI), 6754 (YK KVQNLCG DDTADHAVNI VG) and 6762 (NEVSERVHVYHILKHIKDGK), were determined by means of competition assays with high-binding peptide glycine analogues. The identification of peptides which bind to erythrocyte membrane is important in understanding the process of RBC invasion by P. falciparum merozoites.

Development and Application of Surface Plasmon Resonance-Based Biosensors for the Detection of Cell–Ligand Interactions

Surface plasmon resonance (SPR)-based biosensors were investigated with a view to providing a portable, inexpensive alternative to existing technologies for “real-time” biomolecular interaction analysis of whole cell–ligand interactions. A fiber optic SPR-based (FOSPR) biosensor, employing wavelength-dependent SPR, was constructed to enable continuous real-time data acquisition. In addition, a commercially available integrated angle-dependent SPR-based refractometer (ISPR) was modified to facilitate biosensing applications. Solid-phase detection of whole red blood cells (RBCs) using affinity-captured blood group specific antibodies was demonstrated using the BIACORE 1000, BIACORE Probe, FOSPR, and ISPR sensors. Nonspecific binding of RBCs to the hydrogel-based biointerface was negligible. However, the background noise level of the FOSPR-based biosensor was ∼25-fold higher than that of the widely used BIACORE 1000 system while that of the ISPR-based biosensor was over 100-fold higher. Nevertheless, the FOSPR biosensor was suitable for the analysis of macromolecular analytes contained in crude matrices.

Plasmodium falciparum: red blood cell binding studies of peptides derived from histidine-rich KAHRP-I, HRP-II and HRP-III proteins

Histidine-rich proteins have been associated with Plasmodium falciparum infected red blood cells (RBC) cytoadherence, and RBC rosetting; these phenomena may cause clogging of the post-capillary venules, this being one of the main causes of severe cerebral malaria. They may also participate in parasite mature stages’ evasion of the immune system and their subsequent destruction in the spleen. Non-overlapping synthetic peptides, corresponding to entire amino acid sequences reported for the KAHRP-I, HRP-II and HRP-III proteins, were used in RBC binding assays. Peptides with high and low binding activity were recognized. The KAHRP-I protein shows 3 peptides with high binding affinity to RBCs, two of them variable (peptide 6783, sequence 321QNYVHPWSGYSAPYGVPHGA 340 and peptide 6789, sequence 441KKREKSIMEKNHAAKKLTKK 460) and the other conserved (peptide 6786, sequence 381KSKKHKDHDGEKKKSKKHKD 400) having affinity constant of around 190 nM and 1000 binding sites per cell. Interestingly, this peptide shares aminoacid sequences with one reported as being recognized by malaria exposed human antibodies. The HRP-I protein also presents one conserved peptide (peptide 6800, sequence 24NNSAFNNNLCSKNAKGLNLN 43) with high affinity, located in the amino terminal region of the native protein, having 210 nM affinity constant and 6000 receptor sites. The HRP-III protein only contains peptides with low binding activity. Treatment of red blood cells with neuraminidase reduces the binding of the conserved high binding 6786 and 6800 peptides. Anti-glycophorins A, B and C antibodies inhibit the binding of the conserved high binding 6786 and 6800 peptides. Furthermore, the specific determination of glycoproteins by chemioluminescenoe, in SDS/PAGE western blot, suggests that these glycophorins could be the receptor for these high binding peptides. High binding peptides’ critical amino acids, involved in RBC binding were determined by means of competition binding assays.

Technetium-99m ethylene dicysteine: a new renal tubular function agent.

Technetium-99m ethylene dicysteine (EC), a metabolite of ethylene cysteine dimer (ECD), is a new technetium-labelled renal tubular function tracer introduced as an alternative to ortho-iodohippurate (OIH) and with imaging qualities similar to 99mTc-mercaptoacetyltriglycine (MAG3). The elimination of 99mTc-EC is principally via active tubular transport. It is available in lyophilised kit form which can be easily prepared at room temperature, and the compound remains stable for at least 8 h. Both in normal individuals and in patients, plasma clearance of 99mTc-EC has been reported to be around 0.75 of OIH clearance. Thus there is a very strict correlation between 99mTc-EC and OIH clearance, and several algorithms are available to estimate OIH clearance from 99mTc-EC clearance. The renal extraction ratio of 99mTc-EC is 0.70. The distribution volume of 99mTc-EC is twice that of 99mTc-MAG3 (20% of body weight) and slightly higher than that of OIH. The plasma protein-bound fraction of 99mTc-EC (30%) is significantly lower than that of 99mTc-MAG3 and OIH. The same applies to red blood cell binding of 99mTc-EC (5.7%). There is negligible uptake in the liver and intestines. Within 1 h 70% of 99mTc-EC is excreted in the urine. 99mTc-EC provides the same scintigraphic information as 99mTc-MAG3. The lower liver activity makes 99mTc-EC particularly attractive in patients with renal failure. The 99mTc-EC clearance can be accurately estimated from a single plasma sample obtained at 54 min after injection. In conclusion, 99mTc-EC is a suitable renal imaging agent and for some applications is even more attractive than OIH: it provides an index of tubular function and yields high-quality images. The labelling procedure is easy, radiochemical purity is high and the complex is stable for a long time. The extent to which 99mTc-EC is adopted for clinical use will ultimately depend upon its cost and availability.

A sulfated chitin inhibits hemagglutination by Theileria sergenti merozoites

Five structurally different polysaccharides were examined for their hemagglutination inhibition activity. A (1→4)-linked homopolysaccharide composed of N-acetyl, 6- O-carboxymethyl glucosamine (CM-chitin) showed no inhibitory activity but a 3,6- O-sulfated derivative of CM chitin (SCM-chitin III) showed potent hemagglutination inhibition activity. A N-sulfated derivative of N-deacetylated CM chitin (SCM-chitosan) however, was not effective. Chondroitin sulfate and dermatan sulfate, both of which consisted of disaccharide repeats of (1→3)-linked uronic acid and galactosamine sugar residues (→4GlcAb1/IdoAa1→3GalNAcb1), did not interfere with the red blood cell (RBC) binding by T. sergenti merozoite. Thus, the sulfated glycoconjugate interaction is presumed to be an important process in the RBC invasion by the Theilerial merozoite.

The Myeloid-specific Sialic Acid-binding Receptor, CD33, Associates with the Protein-tyrosine Phosphatases, SHP-1 and SHP-2

The myeloid restricted membrane glycoprotein, CD33, is a member of the recently characterized "sialic acid-binding immunoglobulin-related lectin" family. Although CD33 can mediate sialic acid-dependent cell interactions as a recombinant protein, its function in myeloid cells has yet to be determined. Since CD33 contains two potential immunoreceptor tyrosine-based inhibition motifs in its cytoplasmic tail, we investigated whether it might act as a signaling receptor in myeloid cells. Tyrosine phosphorylation of CD33 in myeloid cell lines was stimulated by cell surface cross-linking or by pervanadate, and inhibited by PP2, a specific inhibitor of Src family tyrosine kinases. Phosphorylated CD33 recruited both the protein-tyrosine phosphatases, SHP-1 and SHP-2. CD33 was dephosphorylated in vitro by the co-immunoprecipitated tyrosine phosphatases, suggesting that it might also be an in vivo substrate. The first CD33 phosphotyrosine motif is dominant in CD33-SHP-1/SHP-2 interactions, since mutating tyrosine 340 in a CD33-cytoplasmic tail fusion protein significantly reduced binding to SHP-1 and SHP-2 in THP-1 lysates, while mutation of tyrosine 358 had no effect. Furthermore, the NH2-terminal Src homology 2 domain of SHP-1 and SHP-2, believed to be essential for phosphatase activation, selectively bound a CD33 phosphopeptide containing tyrosine 340 but not one containing tyrosine 358. Finally, mutation of tyrosine 340 increased red blood cell binding by CD33 expressed in COS cells. Hence, CD33 signaling through selective recruitment of SHP-1/SHP-2 may modulate its ligand(s) binding activity.

A Toxoplasma Lectin-like Activity Specific for Sulfated Polysaccharides Is Involved in Host Cell Infection

Toxoplasma gondii is one of the most widespread parasites of humans and animals. The parasite has a remarkable ability to invade a broad range of cells within its mammalian hosts by mechanisms that are poorly understood at the molecular level. This broad host cell specificity suggests that adhesion should involve the recognition of ubiquitous surface-exposed host molecules or, alternatively, the presence of various parasite attachment molecules able to recognize different host cell receptors. We have discovered a sugar-binding activity (lectin) in tachyzoites of T. gondii that plays a role in vitro in erythrocyte agglutination and infection of human fibroblasts and epithelial cells. The ability to agglutinate erythrocytes can be reversed by a variety of soluble glycoconjugates, of which heparin, fucoidan, and dextran sulfate were the most effective. Interestingly, infectivity of tachyzoites for human foreskin fibroblasts, cells that are commonly used to grow T. gondii in vitro, was increased by low concentrations of the sulfated glycoconjugates that inhibited hemagglutination activity (i.e. dextran sulfate and fucoidan) whereas high concentrations inhibited parasite infection. Furthermore, inhibition of glycosaminoglycan biosynthesis and sulfation on the host cells reduced Toxoplasma infectivity. Finally, Toxoplasma tachyzoites showed a reduced ability to infect epithelial cell mutants deficient in the biosynthesis of surface proteoglycans. The probable identity of the hemagglutinin(s) was investigated by 1) direct binding of red blood cells to filter blots of Toxoplasma proteins separated by polyacrylamide gel electrophoresis, and 2) binding of metabolically labeled parasite proteins to fixed mammalian cells. Three parasite bands were thus identified as candidate adhesins. These results suggest that attachment of T. gondii to its target cell is mediated by parasite lectins and that sulfated sugars on the surface of host cells may function as a key parasite receptor.

Plasmodium Falciparum-infected Erythrocytes: A Mutational Analysis of Cytoadherence via Murine Thrombomodulin

The pathophysiologic events leading to organ damage in Plasmodium falciparum malaria infections involve adhesion and sequestration of parasite-infected erythrocytes (PRBC) to the vascular endothelium and syncytiotrophoblast. Several potential receptors to which the PRBCs may bind have recently been identified, one of which is thrombomodulin (TM). TM has been implicated particularly in mediating sequestration of P. falciparum-infected erythrocytes in the placenta and brain, two sites of disease associated with high morbidity. In order to establish that binding of parasite-infected red blood cells to TM is dependent on its containing chondroitin-4-sulfate (CSA), we have mutated the CSA-attachment site of murine TM, and expressed this mutant form (TMsergly) in COS-7 cells. In cytoadhesion assays, we demonstrate that, in contrast to wild-type TM which contains CSA and supports the adhesion of 1466 PRBCs/mm2, TMser-gly does not contain CSA and adhesion of PRBCs to those cells expressing TMser-gly is entirely abrogated (200 PRBCs/mm2). These studies further confirm that the CSA of TM may play a role in the pathophysiology of malaria by providing a binding site for PRBCs.

Lectin-like oxidized low-density lipoprotein receptor 1 mediates phagocytosis of aged/apoptotic cells in endothelial cells.

Recognition of the exposure of phosphatidylserine (PS) on the outer surface of plasma membrane has been implicated in the phagocytosis of aged/apoptotic cells. Because oxidized low-density lipoprotein (OxLDL) has been reported to block the phagocytosis, here we examined whether lectin-like OxLDL receptor 1 (LOX-1), the OxLDL receptor in endothelial cells, mediates phagocytosis of aged/apoptotic cells by endothelial cells. Cultured bovine aortic endothelial cells (BAE) and Chinese hamster ovary (CHO) cells expressing bovine LOX-1 (BLOX-1-CHO), but not wild-type CHO-K1 cells, bound aged red blood cells (RBC) and apoptotic cells, which were further phagocytosed. The binding of aged RBC and the phagocytosis of apoptotic cells were inhibited by OxLDL, acetyl LDL, and other LOX-1 ligands in both BAE and BLOX-1-CHO. mAb against LOX-1 blocked the binding of aged RBC to BAE, suggesting a role for LOX-1 in the recognition of aged cells. The recombinant soluble LOX-1 inhibited the interactions of aged/apoptotic cells with both BLOX-1-CHO and BAE and distinguished aged RBC from native RBC and apoptotic cells from native cells. PS liposome inhibited these LOX-1-mediated interactions with aged/apoptotic cells, suggesting LOX-1 recognizes PS of the apoptotic cells. PS exposed on the surface of apoptotic cells is known to be procoagulant. Accordingly, increased OxLDL may be one of the reasons for enhanced coagulation in atherosclerosis, inhibiting the removal of apoptotic cells.

Pharmacology and toxicology of artelinic acid: preclinical investigations on pharmacokinetics, metabolism, protein and red blood cell binding, and acute and anorectic toxicities

The pharmacokinetics, metabolism, protein binding, red blood cell (RBC) binding, stability in vitro, and acute and anorectic toxicity of artelinic acid (ARTL) were investigated in various animal species and human blood samples. Absorption and distribution following 10 mg/kg intramuscular or oral administration in dogs and rats were very rapid with t 1 2 0·12–0·54; there were also a high AUC (11262 ng/h/mL) and V ss (9·5 L/kg), low CL (15 mL/min/kg) and long elimination time ( t 1 2 = 2·6 h ), compared with rat data. Oral bioavailability of ARTL was 79·7% in dogs and 30·1% in rats. The conversion of ARTL to dihydroartemisinin (DART) in dogs (0·1–0·5% of total dose) after 3 routes of administration (intravenous, intramuscular and oral) was 10-fold lower than that in rats. In rats dosed with [ 14C]ARTL, unchanged ARTL accounted for less than 13% of the total radioactivity after all 3 administration routes, suggesting that ARTL was extensively biotransformed. The half-lives of total radioactivity (21–49 h) in urine were much longer than that of unchanged ARTL in plasma (1·4–3·7 h), indicating that some long-lasting metabolites of ARTL were formed in rats. The mass balance data showed that 77–83% of total radioactivity was recovered in urine and faeces. High binding capacity (79–95%) and low binding affinity (1·1–9·3 × 10- 7 m) of ARTL were measured in rat, rabbit, dog, monkey and human plasma. The sol RBC plasma ratios of [ 14C]ARTL were 0·35 and 0·44 for dog and human plasma, respectively. ARTL was much more stable than artesunic acid (ARTS) in rat and dog plasma, and both ARTL and ARTS were more stable in dog plasma than in rat plasma in vitro. The 50% lethal dose (LD 50) of ARTL in rats was about 535 mg/kg. Multiple intramuscular dosing for 7 d of 50 mg/kg/d of ARTL caused mild anorectic toxicity compared to ARTS in rats. In contrast to 4 other artemisinin derivatives, ARTL seems to be a good antimalarial candidate as it has the highest plasma concentration, the highest binding capacities in RBC, the highest oral bioavailability, the longest elimination half-life, the lowest metabolism rate and the lowest toxicity at equivalent dose levels.

Effects of Oxysterols Upon Macrophage and Lymphocyte Functions In Vitro

Oxygenated derivatives of cholesterol (oxysterols), found in high concentrations in atherosclerotic lesions, are potent immunosuppressive agents inhibiting T-cell responses to different stimuli. The action of oxysterols on macrophage functions and macrophage-lymphocyte interaction has been poorly investigated. In this work, the effects of 25-hydroxycholesterol (25-OHCh) and 7-ketocholesterol (7-KCh) upon some functions of murine peritoneal macrophage (PM), such as generation of reactive oxygen intermediates (ROI), secretion of neopterin and interleukin-1 (IL-1)-like activity, Fc-receptor (FcR) activity, and murine and human lymphocyte functions, participating in lymphocyte-macrophage interactions, such as macrophage-activating factor (MAF) and Ia-inducing factor (IaIF) secretion, were studied in vitro. 7-KCh in concentration of 5 μg/mL culture medium only, but not 25-OHCh, significantly inhibited ROI generation by zymosan-stimulated PM. Pretreatment of PM for 22 h with 25-OHCh and 7-KCh led to the decrease of IL-1-like activity secretion. 25-OHCh and 7-KCh inhibited both FcR-dependent binding and phagocytosis of sheep red blood cells (SRBC). Oxysterols did not change both spontaneous and lipopolysaccharide-stimulated secretion of neopterin by PM. 25-OHCh dose-dependently and more efficiently than 7-KCh inhibited murine splenocyte secretion of MAF, which activity was determined by the ability of splenocyte-conditioned medium to stimulate ROI generation in PM. Both 25-OHCh and 7-KCh inhibited significantly proliferative activity of human mixed lymphocyte culture (MLC), as well as lymphocyte secretion of IaIF, which stimulates the expression of HLA antigens in cultured human monocytes. Purified Ch did not alter these parameters. These data showed, that some inflammatory functions of macrophages and lymphocytes may be modified by such environmental conditions as the presence of oxysterols.

PECAM-1/CD31, an endothelial receptor for binding Plasmodium falciparum-infected erythrocytes.

Excessive binding of Plasmodium falciparum-infected red blood cells (pRBCs) to the vascular endothelium (cytoadherence) and to uninfected erythrocytes (rosetting) may lead to occlusion of the microvasculature and thereby contribute directly to the acute pathology of severe human malaria. A number of endothelial receptors have been identified as targets for the pRBCs, including CD36, intercellular adhesion molecule-1 (ICAM-1) and chondroitin-4-sulfate (CSA). In vitro, CD36 is the most frequent target of strains from patients with mild as well as severe P. falciparum malaria, but is expressed at low levels on the cerebral microvasculature and therefore seems unlikely to be involved in the evolution of cerebral disease. Strains of P. falciparum that form rosettes are associated both with the occurrence of cerebral malaria and severe anemia. Here we report that malaria-infected RBCs adhere to platelet/endothelial cell adhesion molecule-1 (PECAM-1/CD31) on the vascular endothelium. pRBCs bind to endothelial cells, to PECAM-1/CD31 transfected cells, and directly to recombinant PECAM-1/CD31 absorbed onto plastic. Soluble PECAM-1/CD31 and monoclonal antibodies specific for the amino-terminal segment of PECAM-1/CD31 (domains 1-4) blocked the binding. Interferon-gamma (IFN-gamma)-essential for the development of cerebral malaria in the mouse-was found to augment adhesion of human pRBCs to PECAM-1/CD31 on endothelial cell monolayers. Our results suggest that PECAM-1/CD31 is a virulence-associated endothelial receptor of P. falciparum-infected RBCs.

Population analysis of the non linear red blood cell partitioning and the concentration‐effect relationship of draflazine following various infusion rates

To investigate the impact of the specific red blood cell binding on the pharmacokinetics and pharmacodynamics of the nucleoside transport inhibitor draflazine after i.v. administration at various infusion rates. It was also aimed to relate the red blood cell (RBC) occupancy of draflazine to the ex vivo measured adenosine breakdown inhibition (ABI). Draflazine was administered to healthy volunteers as a 15-min i.v. infusion of 0.25, 0.5, 1, 1.5 and 2.5 mg immediately followed by an infusion of the same dose over 1 h. Plasma and whole blood concentrations were measured up to 120 h post dose, and were related to the ex vivo measured ABI, serving as a pharmacodynamic endpoint. The capacity-limited specific binding of draflazine to the nucleoside transporter located on the erythrocytes was evaluated by a population approach. The estimate of the population parameter typical value (%CV) of the binding constant Kd and the maximal specific binding capacity (Bmax) was 0.385 (3.5) ng ml-1 plasma and 158 (2.1) ng ml-1 RBC, respectively. The non-specific binding was low. The specific binding to the erythrocytes was a source of non-linearity in the pharmacokinetics of draflazine. The total plasma clearance of draflazine slightly decreased with increasing doses, whereas the total clearance in whole blood increased with increasing doses. The sigmoidal Emax equation was used to relate the plasma and whole blood concentration of draflazine to the ex vivo determined ABI. In plasma, typical values (%CV) of Emax, IC50 and Hill factor were 81.4 (1.9)%, 3.76 (9.3) ng ml-1 and 1.06 (3.4), respectively. The relationship in whole blood was much steeper with population parameter typical values (%CV) of Emax, IC50 and Hill factor of 88.2 (2.0)%, 65.7 (2.8) ng ml-1 and 4.47 (5.5), respectively. The RBC occupancy of draflazine did not coincide with the ex vivo measured ABI. The observed relationship between RBC occupancy and ABI was not directly proportional but similar for all studied infusion schemes. The findings of this study show that the occupancy of the nucleoside transporter by draflazine should be at least 90% in order to inhibit substantially adenosine breakdown in vivo. On the basis of these findings it is suggested that a 15 min infusion of 1 mg draflazine followed by an infusion of 1 mg h-1 could be appropriate in patients undergoing a coronary artery bypass grafting.