Thioguanine versus mercaptopurine for therapy of childhood lymphoblastic leukaemia: a comparison of haematological toxicity and drug metabolite concentrations

Stocks of the International Reference Preparation (IRP) for thromboplastin, human, plain, coded BCT/253 and held by the World Health Organization (WHO) are nearly exhausted and must be replaced. For practical reasons the choice of the replacement candidate was restricted to two available human recombinant preparations which were coded as X/95 and Y/95 and calibrated in an international collaborative study involving 19 laboratories from Europe, Australia, Canada and Argentina. To minimize the differences between routes of calibration, the two candidates were calibrated against the existing WHO-IRP from human, rabbit and bovine origin and the final ISI was the resultant average value. On the basis of predefined criteria (i.e., within- and between-laboratory precision of the calibration and the conformity to the calibration model), X/95 was the preferred candidate. The assigned ISI (SE of the mean) value is 0.940 (0.0060) and the interlaboratory coefficient of variation 4.7%.     

Recombinant human luteinizing hormone: a partial physicochemical, biological and immunological characterization

The aim of this study was to partially characterize the glycoform composition of a recombinant human luteinizing hormone preparation (rhLH; Serono), an early version of the material (LHadi) which is currently being assessed for clinical application. Specifically, the charge (pl) and internal carbohydrate complexity of this rhLH was examined and compared with that of an alternative commercially available form of recombinant LH (Crystal Chem) and a pituitary International Reference Preparation (IRP). All preparations were separated by charge by chromatofocusing them on a pH gradient (7-4) using a 4 ml mono-P column is conjunction with a fast performance liquid chromatography system and by complexity of the oligosaccharide structures using concanavalin A (con-A) lectin affinity chromatography. LH in both the unfractionated and fractionated material was assessed by immunoradiometric assay (IRMA, I-LH) and by the in-vitro Leydig cell bioassay (B-LH). Both assays were calibrated against IRP 80/552. The in-vitro biopotency of the preparations was 18187 (Serono rhLH), 12063 (Crystal Chem rhLH) and 6658 (80/552) IU/mg; biological:immunological ratios were 1.14 (80/552), 1.90 (Crystal Chem rhLH) and 1.99 (Serono rhLH). However, similar qualitative data were obtained by both bioassay and immunoradiometric assay following fractionation, with the median pl of the bioactive LH in the preparations being 5.5 (24% > pH 6), 5.52 (18% > pH 6) and 4.97 (0% > pH 6) for the Serono, Crystal Chem and pituitary preparations respectively. Further all three contain < 1% of the complex carbohydrate structures and between 36-44% and 56-63% of the intermediate and simple forms of bioactive LH. In conclusion, the Serono recombinant LH preparation has a higher in-vitro bioactivity and is more basic than the other two preparations although the complexity of its carbohydrate moities appears to be similar.     

Enhancement of growth hormone bioactivity by zinc in the eluted stain assay system

The effects of ionic zinc (Zn2+) on human (h) GH bioactivity have been examined using a lactogenic bioassay. The potencies of pituitary-derived hGH (IRP 80/505), recombinant 22K hGH (IRP 88/624), pituitary-derived human PRL (IRP 84/500), and a recombinant methionyl 20-kilodalton variant of hGH in the presence of selected concentrations of ZnCl2 were investigated with an eluted stain assay that uses Nb2 rat lymphoma cells. This precise colorimetric bioassay is based upon the reduction of a yellow tetrazolium salt, 3-[4,5-dimethyl-thiazol-2-yl]2,5-di-phenyl-tetrazolium bromide, to its purple formazan by lactogen-activated Nb2 cells. Zinc (6-100 microM) enhanced the bioactivity of low doses (< 0.045 nM) of both pituitary-derived and recombinant 22K hGH, although the magnitude of enhancement was considerably less than might have been anticipated from previous binding studies (13). Higher concentrations of pituitary-derived hGH (> 0.045 nM) were inhibited by Zn2+. The bioactivity of recombinant methionyl 20K hGH was greatly enhanced by zinc (3-100 microM). In contrast to hGH, the bioactivity of hPRL was not potentiated by Zn2+. These discriminatory effects of Zn2+ when stimulating via the lactogenic receptor are in concordance with the results of previous radioligand binding studies (13). The striking enhancement of 20K hGH lactogenic bioactivity was observed at Zn2+ concentrations within the physiological range for normal human serum (5-20 microM).     

Intravenous extended infusion of recombinant human soluble thrombomodulin prevented tissue factor-induced disseminated intravascular coagulation in rats

This study demonstrated that intravenous infusion of recombinant human soluble thrombomodulin (rhs-TM) could inhibit disseminated intravascular coagulation (DIC) caused by 4 hr infusion of tissue factor (TF) in rats. Extended infusion of TF reduced fibrinogen and platelet counts and elevated serum FDP level. Pretreatment and coinfusion of rhs-TM could block changes of these DIC-parameters without prolongation of APTT. Heparin, which is a potent anti-DIC drug, could also inhibit these changes with extra prolongation of APTT and PT. Thus, these results suggest thrombomodulin prevent DIC less bleeding tendency than heparin.     

Prothrombin time derived fibrinogen determination on Sysmex CA-6000

AIM: To evaluate PT derived fibrinogen determinations with reference to the Clauss fibrinogen assay using a Sysmex CA-6000 random access coagulation analyser. METHODS: Samples were analysed from normal subjects (n = 20), patients with renal or liver dysfunction (n = 25), critically ill patients (n = 25), patients receiving oral anticoagulant treatment (n = 50), and patients with a haemoglobinopathy (n = 127). Prothrombin times were performed using two thromboplastins: one derived from rabbit brain (Dade: Thromboplastin IS) and the other from recombinant human tissue factor (Dade: Innovin). Fibrinogen was assayed by the Clauss method using a commercial kit (Dade: Fibrinogen). RESULTS: The relation between Clauss fibrinogen and PT derived fibrinogen was found to be dependent on the patient's clinical group and source of the thromboplastin used. When the data from the above sample groups were pooled there was still a significant difference (p < 0.001) between Clauss fibrinogen and PT derived fibrinogen, irrespective of thromboplastin used. CONCLUSIONS: It is unsafe to use the PT derived fibrinogen for patient monitoring owing to non-uniform variability in response to clinical status and reagent employed; however, it may prove to be a useful screening test in a research environment for estimating fibrinogen levels among defined patient groups.     

Diacerhein blocks iron regulatory protein activation in inflamed human monocytes

Iron Regulatory Proteins (IRPs), by modulating expression of ferritin, which stores excess iron in a non toxic form, and transferrin receptor, which controls iron uptake, are the main controller of cellular iron metabolism. During inflammation, modification of IRP activity may affect iron availability, free radical generation and cytokine gene response in inflammatory cells. In the present study we tested the effect of inflammatory stimuli on IRP function in a human monocytic-macrophagic cell line and the possibility of interfering with these pathways by using an antiinflammatory compound, diacerhein (DAR). IRP activity was enhanced by interferon gamma/lipopolysaccarhide (IFN/LPS), and this effect was consistently counteracted by increasing concentrations of DAR. No direct effect of DAR on IRP activity was found in vitro. However, in vivo, similar IRP activation was achieved by exposing cells to nitric oxide (NO) donors and the LPS/IFN-induced activation of IRP was reversed by NO inhibitors. Interestingly, NO-induced IRP activation was efficiently blocked by DAR. These data show for the first time that a clinically useful antiinflammatory compound, DAR, interferes with IRP activation by NO in inflammed human cells.     

Heparin affinity high-performance liquid chromatography as an alternative to bioassay of CS23 mutein of recombinant human basic fibroblast growth factor

We investigated whether a chemical assay by high-performance liquid chromatography (HPLC) as an alternative to the complicated and time-consuming bioassay for CS23 mutein of recombinant human basic fibroblast growth factor (rhbFGF-CS23) using the fetal bovine heart endothelial cell line ATCC CRL 1395. Physically, chemically or enzymatically denatured rhbFGF-CS23 was subjected to heparin affinity (HA)-HPLC and the bioassay. Good agreement was observed between the results obtained by these two methods. Moreover, HA-HPLC gave much more reproducible results (RSD = 1.9%, n = 6) than the bioassay (RSD = 7.4%, n = 18). HA-HPLC is therefore a simple, accurate and reproducible alternative to the bioassay for quality control and stability studies for rhbFGF-CS23 preparations. HA-HPLC is also considered to be applicable to assays for FGFs which have heparin affinity and biological activity similar to those of the CS23 mutein.     

Education for the nurse of tomorrow: a community-focused curriculum

A competitive enzyme-linked immunoadsorbent assay (ELISA) technique has been developed to facilitate quantitative analysis of the earliest step in the initiation of the extrinsic pathway of coagulation, i.e., complex formation of factor VII/VIIa with tissue factor. The ELISA measures the binding of biotinylated human plasma factor VII to relipidated recombinant human tissue factor. Quantitation of the relative affinity (expressed as IC50) of any factor VII molecular population or structural analogue for tissue factor can be determined by competitive binding. Subnanomolar concentrations of both wild-type recombinant human factor VII (rFVII) and rFVII(R152Q), a mutation at the FVII activation site, competed effectively with biotinylated plasma-derived factor VII in binding to tissue factor. In contrast, the affinity of rFVII(R79Q), a mutation in the first epidermal growth factor-like domain, was 12-fold lower. Following activation of rFVII(R79Q), its affinity for tissue factor and enzymatic activity increased 4-fold and 6-fold, respectively. For wild-type rFVII, enzymatic activity rose significantly following activation. However, its affinity for tissue factor was unchanged. We conclude that both the activation state of factor VII and the mutation of amino-acid residues within the first epidermal growth factor-like domain may alter the affinity of factor VII for tissue factor.     

Expression and biochemical characterization of iron regulatory proteins 1 and 2 in Saccharomyces cerevisiae

Iron-regulatory proteins (IRPs) 1 and 2 are cytosolic RNA-binding proteins that bind to specific stem-loop structures, termed iron-responsive elements (IREs) that are located in the untranslated regions of specific mRNAs encoding proteins involved in iron metabolism. The binding of IRPs to IREs regulates either translation or stabilization of mRNA. Although IRP1 and IRP2 are similar proteins in that they are ubiquitously expressed and are negatively regulated by iron, they are regulated by iron by different mechanisms. IRP1, the well-characterized IRP in cells, is a dual-function protein exhibiting either aconitase activity when cellular iron is abundant or RNA-binding activity when cellular iron is scarce. In contrast, IRP2 lacks detectable aconitase activity and functions exclusively as an RNA-binding protein. To study and compare the biochemical characteristics of IRP1 and IRP2, we expressed wild-type and mutant rat IRP1 and IRP2 in the yeast Saccharomyces cerevisiae. IRP1 and IRP2 expressed in yeast bind the IRE RNA with high affinity, resulting in the inhibition of translation of an IRE-reporter mRNA. Mutant IRP2s lacking a 73 amino acid domain unique to IRP2 and a mutant IRP1 containing an insertion of this domain bound RNA, but lacked detectable aconitase activity, suggesting that the presence of this domain prevents aconitase activity. Like IRP1, the RNA-binding activity of IRP2 was sensitive to inactivation by N-ethylmaleimide (NEM) or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), indicating IRP2 contains a cysteine(s) that is (are) necessary for RNA binding. However, unlike IRP1, where reconstitution of the 4Fe-4S cluster resulted in a loss in RNA-binding activity, the RNA-binding activity of IRP2 was unaffected using the same iron treatment. These data suggested that IRP2 does not contain a 4Fe-4S cluster similar to the cluster in IRP1, indicating that they sense iron by different mechanisms.     

Preferential activation of iron regulatory protein-2 in cell lines as a result of higher sensitivity to iron

Iron regulatory proteins (IRP)-1 and 2 are cytoplasmic mRNA-binding proteins that control intracellular iron homeostasis by regulating the translation of ferritin mRNA and stability of transferrin receptor mRNA in an iron-dependent fashion. Although structurally and functionally similar, the two IRP are different in their mode of regulation, pattern of tissue expression and modulation by multiple factors, such as bioradicals. In the present study RNA bandshift assays demonstrated that IRP-2, but not IRP-1, activity was higher in cultured cells than in tissues. Increased expression of IRP-2 in cell lines was not related to immortalization and differentiation but seemed associated to cell proliferation, although not closely dependent on cell growth rate. As a growing cell consumes more iron than its quiescent counterpart, we assessed the iron status of cell lines and found that ferritin content was lower than in tissues. Analysis of IRP activity in cell lines supplemented with heme or non-heme iron and in livers of iron-loaded and iron-deficient rats indicated that IRP-2 responds more promptly than IRP-1 to modulations of iron content. We propose that enhanced IRP-2 activity in cultured cells could be due to a proliferation-dependent, relative iron deficiency that is sensed first by IRP-2.     

Nerve growth factor from the venom of the Chinese cobra Naja naja atra: purification and description of non-neuronal activities

Nerve growth factor (NGF) was separated from crude Naja naja atra venom by using weak cation-exchange chromatography, followed by reversed-phase liquid chromatography. The yield of the purification was 0.2-0.5% (w/w). The mol. wt was determined to be 13,600 and the protein still induced the typical fibre outgrowth of cultured PC-12 cells in a concentration range of 5-10 ng/ml. Beside this neuronal effect we demonstrated non-neuronal effects of cobra venom NGF, such as induction of plasma extravasation and histamine release from whole blood cells. With human leucocyte preparations, including enriched basophils, there was an increase in C5a-induced histamine release, whereas NGF alone was inactive. Cobra NGF was one-tenth as potent as human recombinant NGF, with a half-maximal stimulation occurring at 10 ng/ml. Cobra NGF and human recombinant NGF showed a modulatory effect on histamine release comparable to the haematopoietic growth factor IL-3. Thus, the non-neuronal effects of cobra NGF may account for immunomodulatory activities during inflammatory events.     

Role of epidermal growth factor receptor and STAT-3 activation in autonomous proliferation of SUM-102PT human breast cancer cells

This report describes the isolation and characterization of a new human breast cancer cell line, SUM-102PT, obtained from a minimally invasive human breast carcinoma. SUM-102PT cells have a near diploid karyotype, and early-passage cells had minor chromosomal abnormalities including a 5, 12 and a 6, 16 reciprocal translocation. The cells were isolated and have been continually cultured in three defined media, one of which contains exogenous epidermal growth factor (EGF). SUM-102PT cells have also been carried in an EGF-free medium supplemented with progesterone. All SUM-102PT cells require EGF receptor (EGFR) activation for continuous growth, because incubation of the cells with EGFR-neutralizing antibodies or with EGFR kinase inhibitors blocks growth of these cells. Southern analysis indicates that the EGFR gene is not amplified in these cells; however, these cells express high levels of EGFR mRNA. Thus, SUM-102PT is representative of a class of human breast cancers characterized by high level EGFR expression in the absence of gene amplification. SUM-102PT cells cultured in EGF-free, progesterone-containing medium express high levels of constitutively active EGFR. Conditioned medium from SUM-102PT cells contains an EGF-like mitogen that binds to a heparin-agarose affinity matrix with high affinity. Northern analysis for various EGF family members indicates that SUM-102PT cells synthesize heparin binding (HB)-EGF mRNA. HB-EGF protein is detectable on the surface of these cells by immunohistochemistry, and SUM-102PT cells are killed by diphtheria toxin, which acts by binding to HB-EGF. Furthermore, HB-EGF antibodies partially neutralize the mitogenic activity of the conditioned medium. Thus, EGFR activation in SUM-102PT cells is mediated, at least in part, by autocrine/juxtacrine stimulation by HB-EGF. SUM-102PT cells also express constitutively active STAT-3 homodimers. Constitutively tyrosine-phosphorylated STAT-3 homodimers were also detected in another breast cancer cell line, MDA468, which has an EGFR amplification and also has constitutive EGFR activity. Thus, SUM-102PT is a new human breast cancer cell line that expresses activated EGFR as a result of an autocrine/juxtacrine interaction with HB-EGF which, in turn, results in activation of STAT-3.     

Recombinant factor VIIa corrects prothrombin time in cirrhotic patients: a preliminary study

BACKGROUND & AIMS: Cirrhotic patients with a prolonged prothrombin time (PT) are known to have low levels of factor VII. Because the current modalities to correct this problem are not ideal, recombinant factor VIIa (rFVIIa) may be useful in correcting the prolonged PT observed in the coagulopathy of cirrhosis. The aim of this study was to evaluate the effectiveness of rFVIIa in nonbleeding volunteer patients with the coagulopathy of cirrhosis. METHODS: A preliminary, single-center, dose-escalation trial was performed. Cirrhotic patients with a PT of > 2 seconds above the upper limit of the reference value received an intramuscular injection of vitamin K. Ten patients whose PT did not correct to within 2 seconds above the control of the upper limit of the reference value were given three successive dosages of rFVIIa (5, 20, and 80 micrograms/kg) during a 3-week period. RESULTS: The mean PT transiently corrected to normal in all three dosage groups. No adverse effects were noted. There was no evidence of the induction of disseminated intravascular coagulation. CONCLUSIONS: This preliminary trial shows rFVIIa to be effective in transiently reversing the prolonged PT in a select group of nonbleeding cirrhotic patients. These preliminary observations support conducting a large-scale efficacy trial.     

Preferential antibody recognition of structurally distinct HIV-1 gp120 molecules

We have developed an assay, using a biosensor matrix and surface plasmon resonance, that rapidly and reproducibly measures antibody reactivity to human immunodeficiency virus type 1 (HIV-1) gp120 in various structural conformations. In particular, antibodies displaying preferential reactivity to a CD4-binding competent ("native," rgp120) or CD4-binding incompetent ("reduced," rcmgp120) monomeric gp120 molecule were distinguished. This technique has advantages over conventional enzyme-linked immunosorbent assay (ELISA) methodology in which it is difficult to control the concentration of protein adsorbed to the ELISA wells and a significant disruption of protein structure occurs on adsorption. A population of gp120 molecules that lacked CD4 receptor binding capacity and bound antibodies specific for reduced gp120 was found in several native gp120 preparations. The relative amount of this CD4-binding incompetent population varied among the various preparations studied. This presence of CD4-binding incompetent molecules within various native recombinant gp120 preparations may have implications for HIV-1 envelope vaccine development. By measuring antibody-binding ratios, several monoclonal antibodies were identified, which, although elicited by immunization with various native gp120 preparations, bound specifically to reduced gp120. The ability to screen antibody specificity against HIV-1 envelope proteins with different conformations will assist in determining the quality of antibodies induced by various HIV-1 envelope vaccine candidates.     

Impaired human tissue factor-mediated activity in blood clotting factor VIINagoya (Arg304-->Trp). Evidence that a region in the catalytic domain of factor VII is important for the association with tissue factor

A 37-year-old man was found to have an inherited abnormal factor VII (FVII) (designated FVIINagoya) that has a lower FVII procoagulant activity when tested in a one-stage assay using rabbit, simian, or human tissue factor (TF), but surprisingly has a nearly normal activity using bovine TF. DNA sequence analysis of the propositus' FVII gene revealed a C to T mutation that results in a novel amino acid substitution of Arg304 to Trp. Restriction enzyme analysis of the amplified fragment obtained from each family member demonstrated that the patient is homozygous for the point mutation, and the mother and sister of the patient are heterozygous for this mutation. To elucidate the abnormality in FVIINagoya, we compared the recombinant mutant FVII with normal FVII. In the presence of human TF, kinetic analyses demonstrated that the apparent Km values of FVIINagoya for factor Xa-mediated FVII activation and for FVIIa-mediated factor X (FX) activation were 3.6- and 4.5-fold higher than those of normal FVII, respectively. The ligand binding assay to human TF also showed that FVIINagoya had a 5.2-fold larger apparent Kd than normal. In the presence of bovine TF, however, these values showed no difference between normal and mutant FVII. These findings indicate that the major abnormality in FVIINagoya is not a catalytic dysfunction but an impaired complex-formation of human TF.FVII.FX, suggesting the importance of Arg304 in the interaction with human TF.     

Pathogenicity and lethality of a minute intestinal fluke, Neodiplostomum seoulense, to various strains of mice

Atopy is a genetically determined disorder that affects 10%-20% of the population. Many symptoms of patients with atopy (allergic rhinitis, conjunctivitis, asthma, and anaphylaxis) result from events occurring after crosslinking of cell-bound IgE by per se innocuous environmental antigens. The frequently raised hypothesis that autosensitization can also be a pathogenetic factor in atopy, gained support by our recent demonstration of IgE antibodies against human proteins in atopic dermatitis patients. To unravel the molecular nature of IgE-defined autoantigens, we used serum IgE from atopic dermatitis patients to screen a human epithelial cDNA expression library. One of the cDNA-encoding IgE-reactive products contained 1501 bp of a 2274 bp open-reading frame finally identified by sequence analysis of two additional cDNA clones resulting from oligonucleotide screening. The IgE-defined autoantigen, designated Hom s 1, exhibited an almost complete sequence identity with a recently described antigen recognized by cytotoxic T cells of a squamous cell carcinoma patient. Purified recombinant Hom s 1 specifically bound IgE from patients with severe atopy. When used as immunogen in rabbits, recombinant Hom s 1 gave rise to an anti-serum that reacted with a cytoplasmic protein exhibiting a broad cellular and tissue reactivity (skin, lung > gastrointestinal tract > muscle, brain) and identified a 55 kDa protein in blotted serum IgE preparations. The attractive possibility remains that the Hom s 1-triggered IgE response contributes to the events resulting in allergic tissue inflammation. If so, the respective recombinant molecule may serve as a paradigmatic tool for the diagnosis and treatment of patients with "intrinsic" atopy.     

Characterisation, calibration and comparison by international collaborative study of international standards for the calibration of therapeutic preparations of FSH

Therapeutic preparations of FSH, used primarily for treatment of infertility, are calibrated by in vivo bioassay against international standards (IS) derived from different sources deemed appropriate to their use according to pharmacopoeial monographs. Menotrophins, which have been used for several decades to treat infertility, have been calibrated against the IS for urinary FSH and LH (ISU) but are now being replaced by highly purified urinary FSH or rDNA-derived FSH (rFSH). The aim of this study was to evaluate two preparations of human rFSH and one preparation of highly purified urinary FSH as candidate WHO IS for bioassay in an international collaborative study by 27 laboratories in 12 countries, and to characterise them in a range of in vitro bioassays and immunoassays. The biological activity of the three candidate standards was confirmed by all laboratories using all assays contributed to the study. Dose-response relationships by in vivo bioassay for any of the candidate standards did not differ significantly from that for the ISU. Dose-response relationships obtained in in vitro bioassays and immunoassays were also broadly similar among these preparations although dose-response lines for some preparations appeared to be non-parallel in some immunoassays. For each of the three candidate IS, estimates of the relative potency in terms of ISU by in vivo bioassay did not differ significantly between laboratories. In contrast estimates by immunoassays and in vitro bioassays showed significant differences between laboratories. Estimates of relative potency of the highly purified candidate IS materials in terms of one another exhibited less inter-laboratory variability than estimates in term of ISU. Each of the candidate standards showed adequate stability to serve as an IS. On the basis of the results of this study rFSH (code 92/642) was established as the first IS for FSH, human, recombinant for bioassay with an assigned unitage of 138 IU per ampoule and urinary FSH (code 92/512) was established as the first IS for FSH, human, urinary (urofollitropin) for bioassay with an assigned unitage of 121 IU per ampoule, based on their respective calibration by in vivo bioassay in terms of ISU. These assignments of unitage maintain continuity of unitage for preparations in therapeutic use and also appear to be consistent with one another.     

Transient impact of hemodialysis on gastric myoelectrical activity of uremic patients

Insulin-like growth factor I action has been implicated in the pathogenesis of many different malignancies, including breast cancer. Insulin-like growth factor I receptors (IGF-IRs) are overexpressed in virtually all breast cancer cell lines, in which they are believed to enhance growth and inhibit apoptosis. In this study, the functional activity of IGF-IRs from normal and malignant human breast tissue was assessed. IGF-IR expression was 14-fold higher in malignant breast tissue than in normal breast tissue. IGF-IR autophosphorylation and kinase activity were 2-4-fold higher in purified receptor preparations from malignant breast tissue as compared to normal breast tissue when normalized for receptor number. This increase in receptor function, coupled with the enhanced receptor expression, amounts to a 40-fold elevation in IGF-IR tyrosine kinase activity in malignant breast tissue. The enhanced receptor autophosphorylation and kinase activity were observed in the absence of hormonal stimulation and seem to result from an alteration in the intrinsic activity of the receptor itself. Protein tyrosine phosphatase activity is also increased in malignant breast tissue. These data suggest that the IGF-IR is an important target for breast cancer therapy.