Wright (a+b−) human erythrocytes and plasmodium falciparum malaria

Summary We find Wr(a+b-) erythrocytes of donor M. Fr., which appear to carry a rare glycophorin A variant, to be fully susceptible to invasion by nine isolates of Plasmodium falciparum. Thus we fail to confirm the previous publication on the refractoriness of these erythrocytes. In addition the serum of donor M. Fr., which is known to contain anti-Wr^b directed against an epitope located on glycophorin A in close proximity to the erythrocyte membrane, was not found to inhibit P. falciparum invasion of blood group 0 Rh^– red blood cells.Despite this, different lines of evidence still indicate that glycophorin A is one of the receptors for erythrocyte invasion by P. falciparum. The Wr^b epitope, however, does not appear to represent a distinct receptor site, which is in contrast to previous suggestions.A preliminary communication of this work has been published recently [9]     

N- and O-glycosylation muteins of recombinant human erythropoietin secreted from BHK-21 cells

Single-site glycomuteins of recombinant human erythropoietin (rhuEpo) were constructed and transiently and stably expressed in BHK-21 cells. The transient expression levels varied among muteins, being highest for mutein rhuEpoGln24 followed by wild-type rhuEpo (rhuEpowt). All other glycomuteins, including rhuEpoGln38, rhuEpoGln83, rhuEpoThr126, and rhuEpoGly126, were secreted at lower levels than rhuEpowt. Muteins expressed in stable cell lines showed similar differences in expression levels. Also each mutein could be affinity-purified from culture supernatants, and was biologically active in vivo. Based on secretion rates from BHK-21 cells, the most potent erythropoietin was rhuEpoGln24. This mutein is also considered to have biologic activities that are superior to rhuEpowt.     

Detection of homologous and heterologous malaria antibodies by application of Plasmodium falciparum merozoite antigen to an ELISA

Sera of patients with anti-plasmodial antibodies determined by indirect fluorescence antibody test (IFAT) were used to evaluate an enzyme immunoassay (IgG ELISA) for determining antibodies to Plasmodium falciparum merozoite antigen. The two tests correlated well. Antibodies to species other than P. falciparum, however, were detected by P. falciparum merozoite antigen to a limited extent only.     

Comparative analysis of the activity and content of different streptokinase preparations.

AIMS: The dosage of fibrinolytic agents such as streptokinase must be controlled carefully to maximize therapeutic activity while avoiding adverse effects. Therefore, the integrity and activity of streptokinase products is likely to be clinically relevant. This study was conducted to compare the in vitro characteristics of different streptokinase preparations. METHODS AND RESULTS: Sixteen streptokinase preparations (three of which were recombinant) were compared in a chromogenic substrate activity assay by native, and reducing, sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE), and N-terminal sequencing. Deficiencies in streptokinase activity were observed in most of the products: only three fulfilled the minimum requirements of the European Pharmacopoeia. These were Icikinase (ICI Pharm Ltd, India, only one of two batches tested), Kabikinase (Pharmacia Upjohn, Sweden), and Streptase (Aventis Behring GmbH, Germany). The remaining products exhibited activities ranging from 20.8 to 86.6% of the label claim. Differences in composition and purity were demonstrated by both native and reducing SDS-PAGE. N-terminal sequencing of the recombinant preparations showed differences compared with the native protein--indeed, for one product, the 15 N-terminal amino acids bore no resemblance to streptokinase. CONCLUSION: There are wide variations in the activity, purity, and composition of the available streptokinase preparations.     

Generation of bispecific monoclonal antibodies for two phase radioimmunotherapy

A two phase radioimmunotherapy based on bispecific MAbs in which one arm recognises a tumour antigen and the other a radiolabelled chelate, may prove more effective in the treatment of carcinomas than currently available immunotherapies. To establish this system we first showed that penetration into human carcinoma xenografts as well as long term retention of intact MAb outside the carcinoma cells can be obtained. Epitope saturation was not obtained however, despite the large MAb doses injected i.v. for 10 days. We then generated hybridomas producing high avidity anti-metal chelate MAbs (anti-DTPA-Y). These hybridomas were fused with hybridomas producing MAbs against CEA or GIT-mucin, and stable bispecific MAb producing quadromas were obtained. For the anti-GIT-mucin x anti-chelate MAb a purification procedure based on double anti-idiotype affinity chromatography was shown to result in greater than 95% pure bispecific immunoreactive MAb. Comparative in vivo stability studies profiled DTPA-Y as the chelate of choice for in vivo application.     

Isoform number I--a new tool to evaluate the quality of erythropoietin

The overall isoform number I, calculated from capillary zone electrophoresis (CZE) data, is introduced as a new parameter that enables the assessment of the quality of erythropoietin (EPO) in a simple, yet efficient manner. I is defined as the sum of the products of the individual CZE peak area percent shares (pn) of the EPO isoforms (n = 1-8) and the corresponding individual isoform numbers (n): I=p1x1+p2x2+p3x3+ p4x4+p5x5+p6x6+p7x7+p8x8 The results from an internal collaborative study were used to calculate I-numbers for 2 successive batches of EPO Biological Reference Preparations (BRPs) established by the European Pharmacopoeia (Ph. Eur.) Commission. Based on the results of 6 participating laboratories each, the I-numbers calculated for batch 1 (BRP1) and candidate batch 2 (cBRP2) were I = 518.7 +/- 3.5 (coefficient of variation (CV) = 0.7 %) and I = 542.4 +/- 2.2 (CV = 0.4 %) respectively. Thus, the I-numbers of the 2 EPO preparations clearly indicated the difference between BRP1 and cBRP2 described in the literature, but in a much more apparent and simple manner. Notably, one of the 7 laboratories participating in the study with cBRP2 yielded an outlier value of I = 525.6, pointing towards a suboptimal CZE analysis, and was therefore excluded from the cBRP2 mean value calculation. It is suggested that the overall isoform number I of erythropoietin provides a new and highly valid quality control measure that makes it possible to ensure the batch-to-batch consistency of the active pharmaceutical ingredient of EPO market products.     

Structure-activity relationship of anthracyclines in vitro

The cytotoxic activities of several natural and semisynthetic anthracyclines against L1210 leukemia and two human colon tumor cells (Colon 4, HT 29) in vitro were examined after short (1 h) and long (7 days) incubation times and correlated with the water/octanol partition coefficients and the DNA-binding affinity of the compounds. Analysis of equation in which cytotoxicity against L1210 (1-h incubation) was parabolically related to the partition coefficient revealed an almost exclusive correlation (r = 0.80) between the cytotoxicity and the parameters, and this correlation was only slightly improved by addition of DNA-binding affinity (r = 0.85). On the other hand, cytotoxic activities displayed after continuous incubation were partially related to both partition coefficients (parabolic dependence) and DNA-binding affinities (linear dependence). In this case the correlation between the activity and partition coefficient (r = 0.67) was significantly improved by addition of DNA-binding affinity (r = 0.90). Similar results were also obtained for human colon tumor cells although the corresponding correlation coefficients were generally of lower value, indicating that cytotoxic activity of anthracyclines against these primary resistant cells may be influenced by additional factors not yet determined.     

Toxic effect of isolated glycophorin a on the in vitro growth of Plasmodium falciparum

Summary We have examined the inhibitory potencies of glycophorin A, a mixture of glycophorins B and C, chymotryptic fragments of GpA, desialylated GpA, alkaliborohydride treated GpA, and the O-linked tetrasaccharide isolated from GpA on the invasion of human red blood cells by synchronous Plasmodium falciparum (strain FCB). 50% inhibition of invasion, as measured by ³H-hypoxanthine incorporation into parasites, was achieved at 14 and 155 M for GpA and GpA-CH1, respectively. We have noticed, however, that isolated GpA exhibits a toxic effect on the intraerythrocytic growth of the parasite whereas the chymotryptic fragment (amino acid residues 1–64 of GpA) does not. Thus the inhibitory potency of isolated GpA during erythrocyte invasion by the merozoite should be regarded as the result of both an inhibitory and a toxic effect. The inhibitory effect should be attributed to the carbohydrate-rich outer portion of GpA carrying clusters of neuraminic acid. The toxic effect should be attributed to the hydrophobic region of GpA which might be capable of inserting into the membrane of free merozoites and/or erythrocytes. Our data suggest that results previously obtained with glycoprotein inhibitors carrying hydrophobic portions may have to be questioned.A preliminary report of this subject has been presented at the 1986 Annual Meeting of the Society for Complex Carbohydrates, November 5–7, 1986, Charleston, South Carolina, USA