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R Savino

Publications and source records attributed to R Savino.

At least 37 records · Page 2Linked to original sources
PubMed1996

Interleukin 6 receptor superantagonists are potent inducers of human multiple myeloma cell death.

Interleukin-6 (IL-6) plays a central role in the pathogenesis of multiple myeloma, acting as both a growth and a survival factor for myeloma cells. A series of IL-6 receptor antagonists that are IL-6 variants has been recently obtained, the affinity of which for the ligand-specific receptor chain IL-6R alpha has been maintained or even increased, but the signaling of which is impaired by not being able to bind and/or dimerize the signaling chain gp130. Although IL-6 antagonists have been shown to inhibit the growth of IL-6-dependent myeloma, no information has been gathered on their ability to induce myeloma cell death. We show here that IL-6 receptor antagonists are pro-apoptotic factors for the IL-6-dependent human myeloma cell line XG-1. Their capacity to induce cell death is in direct relation to their affinity for IL-6R alpha, degree of gp130 binding impairment, and efficiency to inhibit intracellular signaling events. Interestingly, the most potent pro-apoptotic molecule, Sant7, counteracts the protective autocrine effect exercised by the limited amounts of IL-6 produced by XG-1 cells and is thus able to induce cell death at higher rate than just IL-6 deprivation. These findings are particularly relevant for the therapy of multiple myeloma.

Antigens, CD↗
PubMed1996

Human interleukin-6 receptor super-antagonists with high potency and wide spectrum on multiple myeloma cells.

Interleukin-6 (IL-6) is the major growth factor for myeloma cells and is believed to participate in the pathogenesis of chronic autoimmune diseases and postmenopausal osteoporosis. IL-6 has been recently shown to possess three topologically distinct receptor binding sites: site 1 for binding to the subunit specific chain IL-6R alpha and sites 2 and 3 for the interaction with two subunits of the signaling chain gp130. We have generated a set of IL-6 variants that behave as potent cytokine receptor super-antagonists carrying substitutions that abolish interaction with gp130 at either site 2 alone (site 2 antagonist) or at both sites 2 and 3 (site 2 + 3 antagonist). In addition, substitutions have been introduced in site 1 that lead to variable increases in binding for IL-6R alpha up to 70-fold. IL-6 super-antagonists inhibit wild-type cytokine activity with efficacy proportional to the increase in receptor binding on a variety of human call lines of different origin, and the most potent molecules display full antagonism at low molar excess to wild-type IL-6. When tested on a representative set of IL-6-dependent human myeloma cell lines, although site 2 super-antagonists were in general quite effective, only the site 2 + 3 antagonist Sant7 showed antagonism on the full spectrum of cells tested. In conclusion, IL-6 super-antagonists are a useful tool for the study of myeloma in vitro and might constitute, in particular Sant7, effective IL-6 blocking agents in vivo.

Antigens, CD↗
PubMed1995

Monovalent phage display of human interleukin (hIL)-6: selection of superbinder variants from a complex molecular repertoire in the hIL-6 D-helix.

Phage display of proteins can be used to study ligand-receptor interaction and for the affinity-maturation of binding sites in polypeptide hormones and/or cytokines. We have expressed human interleukin-6 (hIL-6) on M13 phage in a monovalent fashion as a fusion protein with the phage coat protein, pIII. Phage-displayed hIL-6 is correctly folded, as judged by its ability to interact with conformation-specific anti-hIL-6 monoclonal antibodies (mAb) and with the hIL-6 receptor complex in vitro. We set up an experimental protocol for the efficient affinity selection of hIL-6 phage using the extracellular portion of the hIL-6 receptor alpha (hIL-6R alpha) fixed on a solid phase. This system was used to affinity-purify from a library of hIL-6 variants, in which four residues in the predicted D-helix of the cytokine were fully randomized, mutants binding hIL-6R alpha with higher efficiency than the wild type. When the best-binder variant Q175I/Q183A was combined with a previously identified superbinder S176R [Savino et al., Proc. Natl. Acad. Sci. 90 (1993) 4067-4071], a triple-substitution mutant Q175I/S176R/Q183A (hIL-6IRA) was obtained with a fivefold increased hIL-6R alpha binding and a 2.5-fold enhanced biological activity.

Antibodies, Monoclonal↗
PubMed1995

Definition of a composite binding site for gp130 in human interleukin-6.

The helical cytokine interleukin-6 (IL-6) assembles a multiprotein receptor complex. The starting event in the activation of intracellular signaling is the binding of the IL-6/IL-6R alpha subcomplex to two gp130 chains. The homodimerization of gp130 is triggered by two distinct and independent regions of IL-6 called sites 2 and 3. Several IL-6 antagonists have been obtained that affect signaling, but not IL-6 IL-6R alpha subcomplex formation. In this paper, we analyze in detail the impact of these antagonists on gp130 binding and dimerization and show that each signaling variant affects gp130 dimerization in vitro and that biological activity on cells decreases in precise parallel to the decrease in gp130 dimerization in vitro. All IL-6 antagonists can be classified into two groups, mapping at either site 2 or 3 in correspondence to their mode of interaction with gp130. We found that site 3 is a large region, which includes residues at the beginning of helix D spatially flanked by residues in the putative AB loop and located at one extremity of the cytokine 4-helix bundle. Interestingly, in leukemia inhibitory factor, another cytokine that signals through gp130, site 3, is topologically conserved but has evolved to bind leukemia inhibitory factor receptor.

Antigens, CD↗
PubMed1995

Two distinct and independent sites on IL-6 trigger gp 130 dimer formation and signalling.

The helical cytokine interleukin (IL) 6 and its specific binding subunit IL-6R alpha form a 1:1 complex which, by promoting homodimerization of the signalling subunit gp130 on the surface of target cells, triggers intracellular responses. We expressed differently tagged forms of gp130 and used them in solution-phase binding assays to show that the soluble extracellular domains of gp130 undergo dimerization in the absence of membranes. In vitro receptor assembly reactions were also performed in the presence of two sets of IL-6 variants carrying amino acid substitutions in two distinct areas of the cytokine surface (site 2, comprising exposed residues in the A and C helices, and site 3, in the terminal part of the CD loop). The binding affinity to IL-6R alpha of these variants is normal but their biological activity is poor or absent. We demonstrate here that both the site 2 and site 3 IL-6 variants complexed with IL-6R alpha bind a single gp130 molecule but are unable to dimerize it, whereas the combined site 2/3 variants lose the ability to interact with gp130. The binding properties of these variants in vitro, and the result of using a neutralizing monoclonal antibody directed against site 3, lead to the conclusion that gp130 dimer is formed through direct binding at two independent and differently oriented sites on IL-6. Immunoprecipitation experiments further reveal that the fully assembled receptor complex is composed of two IL-6, two IL-6R alpha and two gp130 molecules. We propose here a model representing the IL-6 receptor complex as hexameric, which might be common to other helical cytokines.

Amino Acid Sequence↗
PubMed1995

Lactate versus bicarbonate on-line hemofiltration: a comparative study.

In this study, 6 chronic anuric uremic patients underwent lactate hemofiltration (HF) with a substitution fluid, prepared in bags, for 3 months and bicarbonate HF, with continuous on-line preparation of the substitution fluid, for 3 months. We determined Na+, Cl-, K+, total calcium (Ca), ionized calcium (Ca2+), phosphate, intact parathormone (I-PTH), pH, PCO2, PO2, and HCO3- in plasma before, during, and after the treatment, as well as Ca, Ca2+, phosphate, and I-PTH in the ultrafiltrate. Moreover, heart rate (HR) and mean arterial pressure (MAP) were continually recorded during the procedure. Furthermore, the patients underwent echocardiographic study before and after the session. In comparison with lactate, bicarbonate HF caused a significant increase in HCO3- and a decrease in K+ plasma concentrations. This was detectable, with lower significance, even 44 h after the end of the treatment. At the end of the long interdialytic period (68 h after the end of the treatment), no significant difference was detectable. pH and PCO2 showed a significant but transient increase during HF. The other parameters investigated did not differ significantly between the two methods.

Adult↗
PubMed1994

Rational design of a receptor super-antagonist of human interleukin-6.

Interleukin-6 (IL-6) is a differentiation and growth factor for a variety of cell types and its excessive production plays a major role in the pathogenesis of multiple myeloma and post-menopausal osteoporosis. IL-6, a four-helix bundle cytokine, is believed to interact sequentially with two transmembrane receptors, the low-affinity IL-6 receptor (IL-6R alpha) and the signal transducer gp130, via distinct binding sites. In this paper we show that combined mutations in the predicted A and C helices, previously suggested to establish contacts with gp130, give rise to variants with no bioactivity but unimpaired binding to IL-6R alpha. These mutants behave as full and selective IL-6 receptor antagonists on a variety of human cell lines. Furthermore, a bifacial mutant was generated (called IL-6 super-antagonist) in which the antagonist mutations were combined with amino acid substitutions in the predicted D helix that increase binding for IL-6R alpha. The IL-6 super-antagonist has no bioactivity, but improved first receptor occupancy and, therefore, fully inhibits the wild-type cytokine at low dosage. The demonstration of functionally independent receptor binding sites on IL-6 suggests that it could be possible to design super-antagonists of other helical cytokines which drive the assembly of structurally related multisubunit receptor complexes.

Carcinoma, Hepatocellular↗
PubMed1994

A fast and sensitive colorimetric assay for IL-6 in hepatoma cells based on the production of a secreted form of alkaline phosphatase (SEAP).

Structure-function studies of cytokines require that simple, sensitive and reliable biological assays are available. A well known property of interleukin-6 (IL-6) is that of being able to induce transcription from several liver-specific promoters in human hepatoma cells. However, the available assays of IL-6 in hepatoma cells, which are either based on the detection of increased expression of endogenous acute phase response genes or on the activation of reporter genes transfected under the control of IL-6 responsive promoters, are not very sensitive and are time consuming. We have established a new assay for IL-6 in hepatoma cells which is based on the transfection of an IL-6 inducible promoter/secreted alkaline phosphatase (SEAP) gene fusion and which measures the inducible production and release of SEAP in the culture medium. SEAP activity is measured with a simple colorimetric assay that requires no cell manipulation, thus allowing a large set of samples to be analysed simultaneously. The CRP/SEAP assay can be used in studies on the structure-function relationships of human IL-6.

Alkaline Phosphatase↗
PubMed1994

Generation of interleukin-6 receptor antagonists by molecular-modeling guided mutagenesis of residues important for gp130 activation.

Interleukin-6 (IL-6) drives the sequential assembly of a receptor complex formed by the IL-6 receptor (IL-6R alpha) and the signal transducing subunit, gp130. A model of human IL-6 (hIL-6) was constructed by homology using the structure of bovine granulocyte colony stimulating factor. The modeled cytokine was predicted to interact sequentially with the cytokine binding domains of IL-6R alpha and gp130 bridging them in a way similar to that of the interaction between growth hormone and its homodimeric receptor. Several residues on helices A and C which were predicted as contact points between IL-6 and gp130 and therefore essential for IL-6 signal transduction, were subjected to site-directed mutagenesis individually or in combined form. Interestingly, while single amino acid changes never produced major alterations in IL-6 bioactivity, a subset of double mutants of Y31 and G35 showed a considerable reduction of biological activity and were selectively impaired from associating with gp130 in binding assays in vitro, while they maintained wild-type affinity towards hIL-6-R alpha. More importantly, we demonstrated the antagonistic effect of mutant Y31D/G35F versus wild-type IL-6.

Amino Acid Sequence↗
PubMed1993

Saturation mutagenesis of the human interleukin 6 receptor-binding site: implications for its three-dimensional structure.

Interleukin 6 is a 184-aa polypeptide postulated to belong to the class of helical cytokines. We built a three-dimensional model of human interleukin 6 based on the similarity of its hydrophobicity pattern with that of other cytokines and on the x-ray structure of growth hormone, interleukin 2, interleukin 4, interferon beta, and granulocyte-macrophage colony-stimulating factor. The resulting model is a bundle of four alpha-helices and suggests possible alternative conformations for the 9 C-terminal amino acids; in this region, the importance of Arg-182 and Met-184 for biological activity has been demonstrated [Lutticken, C., Kruttgen, A., Moller, C., Heinrich, P.C. & Rose-John, S. (1991) FEBS Lett. 282, 265-267]. Therefore, we generated a large collection of single-amino acid variants in residues 175-181. Analysis of their biological activity in two systems and the receptor binding properties of a subset of the mutants indicates that the entire region is involved in forming the receptor binding surface and supports the hypothesis that this region does not assume an alpha-helical conformation. Remarkably, we also found a mutant with receptor affinity and biological activity much higher than wild type; the potential therapeutical value of this finding is discussed.

Amino Acid Sequence↗
PubMed1993

Involvement of the Arg179 in the active site of human IL-6.

Three internal-amino acid deletions of amino acids 171-179 of human interleukin 6 (IL-6) were introduced at the cDNA level. While all deletion proteins were biologically inactive, immunoprecipitations with a set of conformation-specific anti-(IL-6) monoclonal antibodies showed that only mutant delta 177-179 does not present major alterations in folding. This finding, together with the observation that delta 177-179 is not able to compete with IL-6 for binding to the soluble human IL-6 receptor, suggested that some or all of these three residues participate to the composition of the receptor-binding site of human IL-6. A large number of single-amino-acid-substitution mutants were generated in residues 177, 178 and 179. Their detailed analysis revealed that Arg179 is crucial for activity in mouse cells, because all amino acid substitutions in this position cause a dramatic drop of biological activity on murine hybridoma cells without affecting the overall protein folding. The only substitution which preserved some residual activity was the conservative Arg to Lys change. This demonstrates the absolute requirement for a positive charge in position 179 for the interaction of human IL-6 with its receptor.

Arginine↗
PubMed1992

Genes for Xenopus laevis U3 small nuclear RNA.

Genomic Southern blots showed there are only 14 to 20 copies of U3 snRNA genes per somatic genome in Xenopus laevis, unlike the highly repetitive, tandem arrangement of other snRNA genes in this organism. Sequencing of two U3 snRNA genes from lambda clones of a genomic library revealed striking similarity upstream, but much more divergence downstream. Consensus motifs common to other U snRNA genes were also found: a distal sequence element (DSE, octamer motif at -222 to -215), a proximal sequence element (PSE, at -62 to -52) and a 3' Box (15 or 16 bp downstream of the U3 genes). The DSE of mammals also has an inverted CCAAT motif specific for U3 snRNA genes, and we find this is conserved in the amphibian U3 snRNA genes. The Xenopus inverted CCAAT motif is exactly one helical turn further upstream of the octamer motif than its mammalian counterpart, suggesting interaction of putative transcription factors bound to these motifs. Mutation of the inverted CCAAT motif and part of an adjacent Sp1 site greatly depresses transcription of the mutant U3 snRNA gene in Xenopus oocytes, implying a role in transcriptional efficiency. Electrophoretic mobility shift assays implicate transcription factor binding to this region.

Animals↗
PubMed1992

Short time treatment with high-efficiency paired filtration dialysis for chronic renal failure.

With the aim of treating with short sessions even chronic uremic patients with cardiovascular instability, we have developed our previous findings about paired filtration dialysis (hemofiltration-hemodialysis in series) by a method we call high-efficiency paired filtration dialysis (HEPFD). We have treated with this method for 6 months 6 chronic anuric uremic patients previously treated for 6 months with hemofiltration (HF) because of their cardiovascular instability. By comparison with HF, HEPFD shows much higher clearances of small molecules with consequent significant decrease of their predialytic values, improvement of Kt/V value, lower beta 2-microglobulin clearance (its predialytic value remains unchanged), and the same tolerability. HEPFD allows short, efficient, and well-tolerated treatments even in patients with cardiovascular instability.

Adult↗
PubMed1992

Plasma lipids and fatty acid levels in chronically uremic patients undergoing blood purification with different methods.

Levels of plasma lipids and the fatty acid composition of major plasma lipid classes have been determined in chronically uremic patients before and after treatments with hemofiltration, high efficiency paired filtration dialysis, or acetate-free biofiltration. The major findings are a decrease of triglycerides and an increase of total free fatty acids (FFAs) at the end of the dialytic session--already reported in the literature--that do not appear to be strictly dependent on heparin administration. The changes in the plasma concentrations of selected saturated and polyunsaturated fatty acids, which may contribute to alteration of functional parameters in the cardiovascular system in dialyzed patients, are different in the various types of techniques.

Adult↗
PubMed1991

Preribosomal RNA processing in Xenopus oocytes does not include cleavage within the external transcribed spacer as an early step.

Recently it has been reported that U3 snRNA is necessary for: (a) internal cleavage at +651/+657 within the external transcribed spacer (ETS) of mouse precursor ribosomal RNA (pre-rRNA); and (b) cleavage at the 5' end of 5.8S rRNA in Xenopus oocytes. To study if U3 snRNA plays a role at more than one processing site in the same system, we have investigated whether internal cleavage sites exist within the ETS of Xenopus oocyte pre-rRNA. The ETS of Xenopus pre-rRNA contains the consensus sequence for the mammalian early processing site (+651/+657 in mouse pre-rRNA), but freshly prepared RNA from Xenopus oocytes has no cuts in this region. The only putative cleavage sites we found in the ETS of Xenopus oocyte pre-rRNA are a cluster further downstream of the mouse early processing site consensus sequence. This cluster is not homologous to the mouse +651/+657 sites because unlike the latter it is (a) not abolished by disruption of U3 snRNA, (b) not cleaved during early steps of pre-rRNA processing, and (c) lacks sequence similarity to the +651/+657 consensus. Therefore, pre-rRNA of Xenopus oocytes does not cleave within the ETS as an early step in rRNA processing. We conclude that cleavage within the ETS is not an obligatory early step needed for the rest of rRNA maturation.

Animals↗
PubMed1990

In vivo disruption of Xenopus U3 snRNA affects ribosomal RNA processing.

DNA oligonucleotide complementary to sequences in the 5' third of U3 snRNA were injected into Xenopus oocyte nuclei to disrupt endogenous U3 snRNA. The effect of this treatment on rRNA processing was examined. We found that some toads have a single rRNA processing pathway, whereas in other toads, two rRNA processing pathways can coexist in a single oocyte. U3 snRNA disruption in toads with the single rRNA processing pathway caused a reduction in 20S and '32S' pre-rRNA. In addition, in toads with two rRNA processing pathways, an increase in '36S' pre-rRNA of the second pathway is observed. This is the first in vivo demonstration that U3 snRNA plays a role in rRNA processing. Cleavage site #3 is at the boundary of ITS 1 and 5.8S and links all of the affected rRNA intermediates: 20S and '32S' are the products of site #3 cleavage in the first pathway and '36S' is the substrate for cleavage at site #3 in the second pathway. We postulate that U3 snRNP folds pre-rRNA into a conformation dictating correct cleavage at processing site #3.

Animals↗