Analysis of autoantibody epitopes on steroid 21-hydroxylase using a panel of monoclonal antibodies

A panel of five mouse monoclonal antibodies (MAbs) to human recombinant steroid 21-hydroxylase (21-OH) were produced, characterized, and used to study the interaction of 21-OH autoantibodies (AAbs) with different epitopes on human 21-OH. AAbs in patients with isolated autoimmune Addison's disease, autoimmune polyglandular syndromes types I and II, and 21-OH antibody-positive patients without overt Addison's disease (25 patients in total) were studied. Four MAbs were IgG1 subclass, one was IgG2a, and all had kappa light chains. The affinities of four of the antibodies were in the range 2.0 x 10(8) M(-1) to 7.0 x 10(8) M(-1), and the affinity of the other was 2.3 x 10(7) M(-1) 21-OH MAbs did not cross-react with 17alpha-hydroxylase (17alpha-OH)) or P450 side chain cleavage enzyme. Studies using a series of 21-OH fragments allowed the identification of short stretches of amino acids (AA) that were involved in forming the MAb binding sites. AA 391-405, defined as epitope region (ER) 1, were found to be important for binding of M21-OH1 and M21-OH2, AA 406-411 (ER2) were important for M21-OH3 and M21-OH4 binding, and AA 335-339 (ER3) for M21-OH5 binding. In addition, MAb Fab or F(ab')2 fragments were used to study 21-OH AAb epitopes in competition experiments. These investigations demonstrated that 21-OH AAbs recognize similar epitopes to the MAbs, with ER2 and ER3 being part of two distinct major epitopes, and ER 1 being part of a minor epitope. Mixtures of M21-OH antibody Fab or F(ab')2 fragments caused almost complete inhibition (80%-95%) of AAb binding in 24 out of 25 sera, and in the case of the remaining serum, the effect was marked but incomplete (67% inhibition). There were no major differences between the binding characteristics of AAbs from patients with different forms of autoimmune adrenal disease. All five 21-OH MAbs reacted with human adrenal tissue in an immunofluorescence test, but only M21-OH1 and M21-OH2 reacted with bovine adrenal tissue in these experiments. None of the MAbs reacted with human ovarian tissue in an immunofluorescence test. Overall, these studies indicate that 21-OH AAbs bind to at least three different epitopes in the C-terminal part of 21-OH, and two of these epitopes appear to be human 21-OH specific.     

Inhibition of vascular endothelial growth factor-induced receptor activation with anti-kinase insert domain-containing receptor single-chain antibodies from a phage display library

A single-chain antibody phage display library was constructed from spleen cells of mice immunized with a soluble form of a human vascular endothelial growth factor (VEGF) receptor, kinase insert domain-containing receptor (KDR). After two rounds of biopanning, >90% of the clones recovered were specifically reactive to KDR. Subsequent selection identified two clones that blocked VEGF binding to KDR. The clones were expressed in Escherichia coli and purified as soluble single-chain Fv (scFv) antibodies. The affinities of the scFv for binding to KDR were determined by BIAcore analysis (2.1 x 10(-9)-5.9 x 10(-9) M). One scFv, p1C11, was shown to inhibit VEGF-induced KDR phosphorylation and VEGF-stimulated DNA synthesis in human umbilical vein endothelial cells. There is much experimental evidence to suggest that the VEGF/KDR/Flk-1 pathway plays an important role in tumor angiogenesis, a process that is essential for tumor growth and metastasis. The antibodies discussed here, which block VEGF binding to KDR, have potential clinical application in the treatment of cancer and other diseases where pathological angiogenesis is involved.     

Efficient in vitro affinity maturation of phage antibodies using BIAcore guided selections

Selection of higher affinity mutant phage antibodies has proven less than straightforward due to sequence dependent differences in phage antibody expression, toxicity to Escherichia coli, and difficulty in eluting the highest affinity phage. These differences lead to selection for increased levels of expression or decreased toxicity rather than for higher affinity. In this work, we demonstrate how surface plasmon resonance as employed in the BIAcore can be used to increase the efficiency of phage antibody selections, yielding greater increments in affinity from a single library. A mutant phage antibody library was created by randomizing nine amino acids located in the V(L) CDR3 of C6.5, a human scFv which binds the tumor antigen c-erbB-2 with a Kd of 1.6 x 10-8 M. The library was subjected to five rounds of selection in solution using decreasing concentrations of biotinylated c-erbB-2. After each round of selection, polyclonal phage were prepared and the rate of binding to c-erbB-2 determined in a BIAcore under mass transport limited conditions. Determination of the rate of binding permitted calculation of the concentration, and hence percent, of binding phage present. Results were used to select the antigen concentration for the next round of selection. To determine the optimal eluent, polyclonal phage was injected in a BIAcore and eluted using one of five different solutions (10 mM HCl, 50 mM HCl, 100 mM HCl, 100 mM triethylamine, 2.6 M MgCl2). Differences were observed in eluent efficacy, which was reflected in significant differences in the affinities of phage antibodies isolated from the library after a round of selection using the different eluents. Use of the BIAcore to determine the optimal eluent and guide the antigen concentration used for selection yielded a C6.5 mutant with a 16 fold reduction in Kd (Kd = 1.0 x 10-9 M). This represents at least a twofold greater increment in affinity than previously obtained from a single library of phage antibodies which bind antigens.     

Characterization of insulin autoantibodies in relatives of patients with type I diabetes

We studied in detail the anti-insulin autoantibodies in 29 nondiabetic relatives of patients with type I diabetes. The affinity of the autoantibodies for [125I]human insulin was high (1.34 x 10(9)-20.71 x 10(9) L/mol), and the capacity was low (0.84 x 10(-12)-37.80 x 10(-12) M). The product of affinity x capacity of each relative's antibodies directly correlated (r = 0.99) with the level of antibodies determined in our standard radioassay. The autoantibodies from each of the subjects studied had the same rank order of affinities for insulin from different species. Guinea pig, fish insulin, and insulin containing Trp rather than Leu in position 13 of the A-chain inhibited minimally the human insulin binding. Human proinsulin, insulin containing Gln rather than Glu in position 17 of the A-chain, and desoctapeptide insulin (des B23-30) all inhibited binding effectively. Insulin autoantibodies in relatives of patients with type I diabetes share common epitope(s), which suggests a common pathogenic mechanism for production of such antibodies. The epitopes from this initial analysis appear to include amino acids B1-B3 and A8-A13. The region recognized can be distinguished from the insulin receptor binding domain.     

p53 gene transfer to the injured rat carotid artery decreases neointimal formation

PURPOSE: We studied the effect of adenovirus-mediated p53 gene transfer on the injured rat carotid artery to determine its ability to decrease the formation of neointima. METHODS: In vivo gene transfer was used in isolated segments of balloon-injured rat carotid arteries. Genetically modified adenovirus containing the gene encoding for wild-type p53 (AdWTp53) was applied in three concentrations: 8 x 10(10), 1.6 x 10(10), and 8 x 10(9) pfu/mL. Control rats received either adenovirus null (AdNull), 8 x 10(10) pfu/mL, or Medium-199 solution (vehicle). Expression of p53 was determined 4 days after gene transfer by Western blotting. Neointimal formation was assessed after 14 days by harvesting carotid arteries and determining the intima/media (I/M) ratio based on cross-sectional area measurement. Simultaneously, immunohistochemistry was done to detect the presence of p53 on smooth muscle cell nuclei. RESULTS: P53 expression was confirmed by Western blotting. There was a significant reduction in neointimal formation on all treated animals compared with controls. The highest dose of AdWTp53 (8 x 10(10) pfu/mL) resulted in a near-total arrest of neointimal formation (I/M = 0.09 +/- 0.03, mean +/- SEM) with P <. 0001 versus vehicle (I/M = 2.23 +/- 0.15) or AdNull (I/M = 2.12 +/-. 12). The intermediate dose of AdWTp53 (1.6 x 10(10) pfu/mL) resulted in an I/M value of 1.04 +/- 0.18, with P <.001 versus vehicle and P =.001 versus AdNull. The lowest dose (8 x 10(9) pfu/mL) resulted in an I/M value of 1.12 +/- 0.18, with P <.001 versus vehicle and P <. 002 versus AdNull. The immunohistochemistry was positive for the presence of p53 in rats infected with AdWTp53. CONCLUSIONS: Adenovirus-mediated gene transfer of p53 protein significantly decreases the formation of neointima in the rat carotid injury model. This may represent a potential therapy for restenosis in humans.     

Sensitive enzyme immunoassay of antibodies to HIV-1 p17 antigen using indirectly immobilized recombinant p17 for diagnosis of HIV-1 infection

Recombinant p17 (rp17) antigen of HIV-1 and maltose binding protein-rp17 fusion protein (MBP-rp17) were immobilized onto polystyrene beads in different ways: rp17 and MBP-rp17 were immobilized directly onto polystyrene beads by physical adsorption; biotinyl-rp17 and biotinyl-MBP-rp17 were immobilized indirectly onto streptavidin-coated polystyrene beads; and 2,4-dinitrophenyl (DNP)-MBP-rp17 was immobilized indirectly onto (anti-DNP) IgG-coated polystyrene beads. These directly and indirectly immobilized antigens were incubated with urine samples containing antibody IgG to p17 antigen and subsequently with rp17-beta-D-galactosidase conjugate or (anti-human IgG gamma-chain) Fab'-beta-D-galactosidase conjugate. Beta-D-galactosidase activity bound to the polystyrene beads was assayed by fluorometry. When rp17-beta-D-galactosidase conjugate was used, signals (fluorescence intensities for bound beta-D-galactosidase activity) were much higher with the indirectly immobilized antigens than those with the directly immobilized antigens. By experiments using (anti-human IgG gamma-chain)Fab'-beta-D-galactosidase conjugate, the binding of rp17-beta-D-galactosidase conjugate to antibodies against p17 antigen bound to directly immobilized rp17 antigen was shown to be seriously limited as compared with that to antibodies against p17 antigen bound to indirectly immobilized DNP-MBP-rp17. When rp17-beta-D-galactosidase conjugate and serum samples were used, serum interference was much less with indirectly immobilized DNP-MBP-rp17 than with directly immobilized rp17 antigen, and the sensitivity of enzyme immunoassay for antibody IgG to p17 antigen using indirectly immobilized DNP-MBP-rp17 was 1,000- to 3,000-fold higher than that of enzyme immunoassay using directly immobilized rp17 antigen and Western blotting for p17 band. This sensitive enzyme immunoassay indicated positivity in HIV-1 seroconversion serum panels as early as conventional methods for antibodies to HIV-1 and earlier than Western blotting for p17 band.     

The soluble extracellular portion of the human interferon-gamma receptor is a valid substitute for evaluating binding characteristics and for neutralizing the biological activity of this cytokine

In order to have a reliable and reproducible source of soluble human interferon-gamma (HuIFN-gamma) receptor at our disposal both for studying binding phenomena and for evaluating its neutralizing potential towards the cytokine, we expressed the extracellular part of the receptor in J558L mouse myeloma cells as a fusion protein with the C-terminal c-myc TAG (HuECR-gamma-TAG). It is expected that the receptor will undergo post-translational modifications comparable to that in humans. The affinity purified soluble receptor was subjected to mass spectrometry analysis resulting in a molecular size of 31 to 40 kDa and showed heterogeneous N-glycosylation with an M(r)-contribution of 4 to 13 kDa. Its HuIFN-gamma binding affinity, determined by real time biospecific interaction (BIAcore) analysis, resulted in a value of Kd = 2 x 10(-9) M, which is in agreement with the high affinity described for the cell anchored complete HuIFN-gamma receptor (Kd = 5-35 x 10(-9) M). HuECR-gamma-TAG was able to neutralize the biological activity of HuIFN-gamma in an in vitro antiviral assay. Furthermore, we report for the first time the association and dissociation rate constants, which were, respectively, 2.4 x 10(5) M-1 s-1 and 4.8 x 10(-4) s-1. In conclusion, this mammalian source of the extracellular soluble HuIFN-gamma receptor represents a valuable tool for extensive in vitro studies of the HuIFN-gamma receptor interaction. Furthermore, in view of its expected low or nonimmunogenicity it opens new ways for immunomodulation in vivo.     

Effects of calcitonin gene-related peptide on wound contraction in denervated and normal rat skin: a preliminary report

The aim of the present study was to observe the effect of calcitonin gene-related peptide (CGRP) on wound contraction in both denervated and normal areas. A total of 100 Wistar rats, each of which had a 2 x 2 cm full-thickness skin defect on the back, were divided into two main control groups and six corresponding experimental groups in which 10 x 10(-9) M and 10 x 10(-12) M synthetic rat CGRP were given intraperitoneally and intradermally. Contraction was assessed weekly with planimetry, and mean surface areas (mm2 +/- SD) of related groups were compared. Complete closure took 4 weeks for the normal control group and 8 weeks for the denervated control group (p < 0.05). The 10 x 10(-12) M CGRP with both types of application showed a decrease in the length of time for complete closure to 3 weeks in the normal experimental groups (p < 0.05), but complete closure still took 4 weeks in normal groups in which 10 x 10(-9) M CGRP was given (p < 0.05). Any dosage of CGRP given intraperitoneally showed no change in the closure period in the denervated experimental groups (p > 0.05). CGRP showed a trophic effect on healing by an increased rate of contraction in the rat model. However, the neural supply to the wound area seemed to be intact because of the necessity of axonal transfer of CGRP.     

Developmental changes in mouse placental cells from several stages of pregnancy in vivo and in vitro

Developmental changes in mouse placentae from the 6th to the 18th day of pregnancy were studied in vivo and in vitro. Placental volume increased from the 6th to the 18th day of pregnancy; however, the total number of cells per placenta reached a plateau on the 14th day. Decidual cells were predominant in the placenta on the 6th day. Placentae obtained from the 10th to the 18th day contained decidual cells, trophoblastic (labyrinth and spongiotrophoblast) cells, and trophoblast giant cells. Decidual cells increased in number from the 6th to the 10th but decreased on the 14th day, whereas trophoblastic cells increased linearly until the 14th day. Two types of placental cells were distinguished in vitro: small fibroblast-like cells and large flattened cells containing 2-3 nuclei. The large cells reacted to anti-desmin antibody, indicating their decidual character. The small cells reacting to anti-keratin antibody appeared to be trophoblastic cells. Decidual cells from all days of gestation were nonproliferative, regressing with time in culture. 17 beta-Estradiol (E, 10(-9) and 10(-8) M), progesterone (P, 10(-10), 10(-9), and 10(-8) M), and a combination of E and P (10(-9) M each) stimulated proliferation of the trophoblastic cells only from the 6th and the 10th days. Keoxifene (2 x 10(-7) M), but not tamoxifen, significantly inhibited the E-induced proliferation of the trophoblastic cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel monoclonal antibodies to putative selectin carbohydrate ligands that inhibit selectin binding to myeloid cells

Four newly developed monoclonal antibodies (MAbs) are characterized using flowcytometry, enzyme-linked immunoadsorbent assay (ELISA), immunoprecipitation and Western blots, carbohydrate epitope mapping, glycosidase cleavage, and competition binding assays. Their effects on selectin binding to myeloid cells was tested. These MAbs react only with myeloid cells. MAbs CI-1, BU60, and HIM95 recognize epitopes expressed by CD11/CD18 (beta2) integrins, while HI247 and CSLEX1 do not. The epitopes require Lewis x [Galbeta1-4 (Fucalpha1-3)GlcNAc] based on reactivity with oligosaccharide-polyacrylamide-biotin or oligosaccharide-BSA conjugates. MAb HI247 recognizes a related structure, sialyl-Lewis x, NeuAcalpha2-3GaLbeta1-4(Fucalpha1-3)GlcNAc. The three MAbs against Lewis x show some minor differences in their reactivity such as recognizing their antigens on CD11/CD18 integrins after endo-beta-galactosidase treatment and recognizing free Lewis x. The hydroxyl group on C-3 of the terminal galactose is important for recognition by MAb CI-1, BU60, and HIM95 as its substitution with sulfo group of sialic acid abolishes the binding of these MAbs. The C-3 sialic acid is crucial for the binding of MAb HI247. Its replacement by sulphate or its cleavage by sialidase eliminates recognition by this MAb. MAbs HI247 and CSLEX-1 did not react in ELISA with immobilized CD11/CD18, suggesting that the majority of sialyl Lewis x on CD11/CD18 molecules may have sialic acid 6-linked rather than 3-linked to galactose. Unexpectedly, MAb BU60 inhibited binding of P-selectin mu chain chimera to HL-60 or U937 cells, while CI-1, HIM95 and three other defined anti-Lewis x MAbs (6C7, M6-1 and LeuM1) did not. MAb HI247 inhibited binding of both E- and P-selectin chimeras to these cell lines more effectively than several characterized MAbs (CSLEX-1, FH6, HECA-452) to sialyl Lewis x and related oligosaccharides. Certain combinations of these anticarbohydrate MAbs had additive inhibitory effects on selectin binding, suggesting a potential application of these new MAbs in cell adhesion/migration and tumor metastasis studies.     

Agonist and receptor binding properties of adrenocorticotropin peptides using the cloned mouse adrenocorticotropin receptor expressed in a stably transfected HeLa cell line

The cloned mouse ACTH receptor was expressed in stably transfected human HeLa cells that lack an endogenous melanocortin receptor. ACTH[1-39] and several N- and C-terminally truncated analogues of ACTH were studied for their ability to stimulate cAMP generation and to displace bound 125I-ACTH. Only three of the peptides tested, ACTH[1-24], ACTH[1-39], and ACTH[1-17] were found to have agonist activity with EC50 values of 7.5, 57, and 49 x 10(-12) M respectively. Two peptides, ACTH[11-24] and ACTH[7-39], were devoid of agonist activity but had substantial competitive antagonist activity with IC50 values of approximately 10(-9) M. In binding studies, ACTH[1-39] and ACTH[1-24] were able to fully displace bound ligand, and Scatchard analysis indicated a dissociation constant (KD) of 0.84 and 0.94 x 10(-9) M for the two peptides, respectively. ACTH[1-17], ACTH[11-24], and ACTH[7-39] were only capable of displacing 60-70% of bound ligand. A three-site model for the interaction of ACTH and its receptor is proposed on the basis of these findings.     

Influence of mass transfer and surface ligand heterogeneity on quantitative BIAcore binding data. Analysis of the interaction of NC10 Fab with an anti-idiotype Fab'

The interaction of monovalent Fab fragments of NC10, an antiviral neuraminidase antibody, and the anti-idiotype antibody 3-2G12 has been used as a model system to demonstrate experimentally the influence of non-ideal binding effects on BIAcore binding data. Because the association rate constant for these two molecules was found to be relatively high (about 5 x 10(5) M-1 S-1), mass transfer was recognised as a potential source of error in the analysis of the interaction kinetics. By manipulation of the flow rate and the surface density of the immobilised ligand, however, the magnitude to this error was minimised. In addition, the application of site-specific immobilisation procedures was found to improve considerably the correlation of experimental binding data to the ideal 1:1 kinetic model such that the discrepancy between experimental and fitted curves was within the noise range of the instrument. Experiments performed to measure the equilibrium constant (KD) in solution resulted in a value of similar magnitude to those obtained from the ratio of the kinetic rate constants, even those measured with a heterogeneous ligand or with a significant mass transfer component. For this system, the experimental complexities introduced by covalent immobilisation did not lead to large errors in the KD values obtained using the BIAcore.     

Comparative evaluation of reactogenicity and immunogenicity of two dosages of oral tetravalent rhesus rotavirus vaccine. US Rhesus Rotavirus Vaccine Study Group

OBJECTIVE: To compare the safety and immunogenicity of two dosages of tetravalent rhesus rotavirus vaccine (RRV-TV) and the effect of age at dosing. METHODS: A total of 195 infants were stratified by age into 2 groups, 6 to 12 weeks and 16 to 24 weeks, and randomly assigned to receive a single dose of placebo or RRV-TV containing either 4 x 10(5) or 4 x 10(6) plaque-forming units (pfu). Symptoms were recorded for 5 days after vaccination. Anti-rotavirus IgA and neutralizing antibody to human rotavirus serotypes G1 to G4 and RRV were measured in serum obtained pre- and postvaccination. RESULTS: Rates of fever > 38 degrees C (9%), diarrhea (6%) and vomiting (8%) were similar in all groups. IgA (69% vs. 49%, P = 0.02) and RRV (85% vs. 66%, P = 0.004) seroconversion rates were significantly higher in the 4 x 10(6) pfu vaccine group as were antibody titers to RRV (440.2 vs. 263.7, P = 0.04). Older infants demonstrated significantly higher seroconversion rates and antibody titers for IgA (71% vs. 52%, P = 0.03; and 110.6 vs. 54.8, P = 0.004) and RRV (92% vs. 66%, P = 0.05 and 498.3 vs. 205.6, P = 0.01) at either dose level than did the younger infants. There were no significant differences in seroconversion rates or antibody titers to human rotavirus types G1 to G4 between the two vaccination groups. CONCLUSIONS: RRV-TV at a dose of 4 x 10(6) pfu can be safely administered to infants 6 to 24 weeks of age. A single dose of 4 x 10(6) pfu of RRV-TV was significantly more immunogenic than a single dose of 4 x 10(5) pfu but did not improve responses to the human serotypes. Older vaccine recipients demonstrated significantly higher IgA and neutralizing antibody seroconversion rates and antibody titers than younger infants independent of dosage.     

Adsorptive stripping voltammetry of bleomycin

In 0.05 M H2SO4 solution, two reductive peaks, P1 and P2, of bleomycin were obtained. The peak potentials E(P1) and E(P2) were -0.83 and -1.09 V (versus Ag/AgCl), respectively. The sensitivity of P2 was much higher than that of P1. The peak current of P2 was proportional to the concentration of bleomycin over the range 1.0 x 10(-9)-1.0 x 10(-7) M with a detection limit of 5.0 x 10(-10) M using adsorptive voltammetry at an accumulation time of 120 s (Ei = -0.80 V). The behaviour of the reduction wave was studied and applied to the determination of bleomycin in mouse serum. The reduction process of P1 was irreversible with adsorptive characteristics and the adsorption behavior obeyed the Frumkin adsorptive isotherm. The adsorptive coefficient beta was 8.9 x 10(5), the interaction factor alpha was 0.94 and the Gibbs energy of adsorption delta G degree was -33.93 kJ mol-1. P2 was an irreversible adsorption peak with catalytic hydrogen properties.     

Ethnic differences in interferon-alpha allele frequencies

We analyzed microsatellite instability (MSI) and loss of heterozygosity (LOH) at 17 microsatellite markers located on chromosomes 2p, 3p, 5q, 6q, 9p, 9q, 17p and 18q in 19 randomly selected keratoacantomas (KAS), in one cutaneous lesion that histologically could not unequivocally be differentiated from squamous cell carcinoma, and in one patient with multiple KAs of longstanding duration. The goals of our study were to determine whether, in a similar manner to some visceral carcinomas, genomic instability could be detected in KAs and to clarify whether molecular analysis might be useful to further characterize KA. MSI was observed in 2 of 21 cases (9.5%) at 5 of 17 loci examined. In one patient with a solitary KA, the presence of MSI and a family history of visceral malignant tumours suggested that the patient might have belonged to a family with Muir-Torre syndrome. In one other MSI+ KA, a definite differential diagnosis in relation to squamous cell carcinoma could not be established. In addition, one sample displayed LOH at 2 of 17 loci analysed whereas in the patient with multiple KAs, LOH at one locus was the only alteration found. In conclusion, the low frequency of MSI and LOH detected in our study suggests that these genetic events are uncommon in KA unless it is associated with a familial disease (e.g. Muir-Torre syndrome) or it has more aggressive histological features.     

Mobilisation of healthy donors with lenograstim and transplantation of HLA-genoidentical blood progenitors in 54 patients with hematological malignancies: a pilot study

Blood cell transplantation (BCT) is now common practice in the autologous setting. We performed a pilot study of allogeneic BCT, collected after the priming of an HLA-identical sibling with a glycosylated rhu-G-CSF (lenograstim) (10 microg/kg). Fifty-four patients were included (38 +/- 11; M/F = 33/21; CML (n = 17), AML (n = 14), ALL (n = 15); MDS (n = 8)). Transplant procedures were standard (TBI regimen = 47 (87%); MTX-CsA: n = 37; CsA-PDN: n = 17). No serious adverse events were reported in donors. A median of 11 (3.5-29.1) x 10(6)/kg CD34+ cells, 332 (33-820) x 10(6)/kg CD3+ cells were collected. Four patients did not engraft (early death: n = 2; graft failure: n = 2). Fifty-one patients initially recovered 0.5 x 10(9)/l ANC and 25 x 10(9)/l platelets at 15 (10-30) and 13 (9-188) days. 29/51 and 29/38 experienced grade > or =2 acute and chronic GVHD. With a median follow-up of 25 months (18-36), relapse rate is 16% +/- 8, survival and DFS probabilities are similar (50% +/- 13). A better outcome is documented for patients under 45 years and in the early phase of the disease (n = 28), with an identical survival and DFS of 71% +/- 13. In conclusion, lenograstim is a potent rhu-G-CSF for mobilisation of allogeneic hematopoietic progenitors. Two-year follow-up indicates good haematological recovery but some concerns about graft failure and chronic GVHD have arisen deserving prospective evaluation.     

Effect of nomegestrol acetate on estrone-sulfatase and 17beta-hydroxysteroid dehydrogenase activities in human breast cancer cells

It is well recognized that estradiol (E2) is one of the most important hormones supporting the growth and evolution of breast cancer. Consequently, to block this hormone before it enters the cancer cell or in the cell itself, has been one of the main targets in recent years. In the present study we explored the effect of the progestin, nomegestrol acetate, on the estrone sulfatase and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activities of MCF-7 and T-47D human breast cancer cells. Using physiological doses of estrone sulfate (E1S: 5 x 10(-9)M), nomegestrol acetate blocked very significantly the conversion of E1S to E2. In the MCF-7 cells, using concentrations of 5 x 10(-6)M and 5 x 10(-5) M of nomegestrol acetate, the decrease of E1S to E2 was, respectively, -43% and -77%. The values were, respectively, -60% and -71% for the T-47D cells. Using E1S at 2 x 10(-6) M and nomegestrol acetate at 10(-5) M, a direct inhibitory effect on the enzyme of -36% and -18% was obtained with the cell homogenate of the MCF-7 and T-47D cells, respectively. In another series of studies, it was observed that after 24 h incubation of a physiological concentration of estrone (E1: 5 x 10(-9)M) this estrogen is converted in a great proportion to E2. Nomegestrol acetate inhibits this transformation by -35% and -85% at 5 x 10(-7)M and 5 x 10(-5)M, respectively in T-47D cells; whereas in the MCF-7 cells the inhibitory effect is only significant, -48%, at 5 x 10(-5)M concentration of nomegestrol acetate. It is concluded that nomegestrol acetate in the hormone-dependent MCF-7 and T-47D breast cancer cells significantly inhibits the estrone sulfatase and 17beta-HSD activities which converts E1S to the biologically active estrogen estradiol. This inhibition provoked by this progestin on the enzymes involved in the biosynthesis of E2 can open new clinical possibilities in breast cancer therapy.     

An immunological interleukine-6 capacitive biosensor using perturbation with a potentiostatic step

An instrument for potentiostatic capacitance measurements, based on perturbation with a potentiostatic step was used. The capacitive sensor consisted of self-assembled sulfur compounds on gold to which antibodies towards Interleukine 6, Il-6, had been immobilized. The biosensor was part of a potentiostatic three-electrode system with an extra reference electrode. Two different methods using epoxy- or carbodiimide coupling of the polyclonal antibodies were compared. The antigen Il-6 could be detected from 5 x 10(-16) to 5 x 10(-13) M with one immobilization method and to more than 5 x 10(-9) M with the other. No labels were necessary since the binding of the antigen was detected directly. The insulating properties of the different layers of the biosensor were investigated.     

Selective inhibition of primary acute myeloid leukaemia cell growth by lovastatin

The insulin receptors from erythrocytes of 50 patients with non-insulin-dependent diabetes mellitus were tested for their ability to autophosphorylate. The assay was performed by a new enzyme-linked immunosorbent assay system that used monoclonal anti-insulin receptor antibodies absorbed to microtiter plates as a first antibody and polyclonal antiphosphotyrosine antibody as a labeled second antibody. By this assay, 3 patients were identified with defects in their insulin receptor kinase, although their defects appeared heterogeneous. Patient 1 had 85% less maximal autophosphorylation with a normal ED50 (1.6 x 10(-9) M insulin). Patient 2, who had polycystic ovary disease, had a 49.2% decrease in maximal autophosphorylation of insulin receptors, and the ED50 was shifted to the right (5.6 x 10(-8) M). Patient 3 with acanthosis nigricans had a normal maximal autophosphorylation, but the ED50 shifted to the right (2.9 x 10(-8) M). The mechanisms for the diversity detected in this assay is not known, but this technique has sufficient specificity and sensitivity to be used to screen for insulin-resistant patients who have a lack of kinase activity.     

Determination of the nucleotide conformation in the productive enzyme-substrate complexes of RNA-depolymerases

Docking of ER-derived vesicles to the cis-Golgi compartment in yeast requires vesicle and target membrane receptors (v-SNAREs and t-SNAREs) and the GTPase Ypt1p. The t-SNARE Sed5p is complexed with Sly1p in vivo. The mutant form Sly1-20p rescues Ypt1p-lacking cells from lethality, suggesting an inhibitory function of Sly1p in v-SNARE/t-SNARE interaction. Using surface plasmon resonance spectroscopy, we found that Sed5p binds Sly1p and Sly1-20p with equally high affinity (K(D) = 5.13 x 10(-9) M and 4.74 x 10(-9) M, respectively). Deletion studies show that the N-terminal half of Sly1p rather than the C-terminus (harbouring the E532K substitution in Sly1-20p) is most critical for its binding to Sed5p. These data appear to argue for an active rather than an inhibitory role of Sly1p in vesicle docking.