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Biomedical subjects

R Laufer

Publications and source records attributed to R Laufer.

At least 19 recordsLinked to original sources
PubMed2006

Development of humanized mice for the study of hepatitis C virus infection.

The development of a small animal model for hepatitis C virus (HCV) infection is a critical issue for the development of novel anti-HCV drugs. To this aim, we have tried many different approaches for generating mice carrying humanized liver. Main efforts were focused on the transplantation of human hepatocytes into immunocompromised mice (SCID-/-, Bg-/-) carrying a genetic lethal liver disease (Alb-uPA). Survival of homozygotic animals should largely depend on early transplantation with healthy hepatocytes. In parallel to establishing a colony of Alb-uPA/SCID/Bg mice, we developed a microsurgical procedure for intrasplenic xenotransplantation of healthy hepatocytes in 1-week-old mice. So far, we generated several chimeras by xenotransplanting human hepatocytes in Alb-uPA+/+/SCID-/-/Bg-/- mice at 1 week after birth. In a first step, identification of successfully engrafted animals is possible by quantification of human serum albumin and human alpha 1 antitrypsin in mouse sera. Additional preliminary histomorphological analysis of liver sections from chimeric animals was also carried out. One of the mice was transiently infected with HCV, reaching viremia levels of approximately 10(5) genomes/mL. However, the efficiency of this system to generate chimeric mice is still very limited. We are currently exploring the use of more robust models of hepatic disease. Moreover, we have been also exploring novel strategies for the generation of chimeric mice by xenotransplanting human adult stem cells, instead of human hepatocytes, at preimmune stages of development.

Animals↗
PubMed2005

Effects of fibrates on human organic anion-transporting polypeptide 1B1-, multidrug resistance protein 2- and P-glycoprotein-mediated transport.

The effects of different fibric acid derivatives (bezafibrate, clofibrate, clofibric acid, fenofibrate, fenofibric acid and gemfibrozil) on human organic anion transporting-polypeptide 1B1 (OATP2, OATP-C, SLC21A6), multidrug resistance protein 2 (MRP2/ABCC2) and MDR1-type P-glycoprotein (P-gp/ABCB1) were examined in vitro. Cyclosporin A (a known inhibitor of OATP1B1 and P-gp), MK-571 (a known inhibitor of MRP2) and cimetidine (an organic cation) were also tested. Bezafibrate, fenofibrate, fenofibric acid and gemfibrozil showed concentration-dependent inhibition of estradiol 17-beta-D-glucuronide uptake by OATP1B1-stably transfected HEK cells, whereas clofibrate and clofibric acid did not show any significant effects up to 100 microM. Inhibition kinetics of gemfibrozil, which exhibited the most significant inhibition on OATP1B1, was shown to be competitive with a Ki = 12.5 microM. None of the fibrates showed any significant inhibition of MRP2-mediated transport, which was evaluated by measuring the uptake of ethacrynic acid glutathione into MRP2-expressing Sf9 membrane vesicles. Only fenofibrate showed moderate P-gp inhibition as assessed by measuring cellular accumulation of vinblastine in a P-gp overexpressing cell-line. Cyclosporin A significantly inhibited OATP1B1 and P-gp, whereas only moderate inhibition was observed on MRP2. The rank order of inhibitory potency of MK-571 was determined as OATP1B1 (IC50: 0.3 microM) > MRP2 (4 microM) > P-gp (25 microM). Cimetidine did not show any effects on these transporters. In conclusion, neither MRP2- nor P-gp-mediated transport is inhibited significantly by the fibrates tested. Considering the plasma protein binding and IC50 values for OATP1B1, only gemfibrozil appeared to have a potential to cause drug-drug interactions by inhibiting OATP1B1 at clinically relevant concentrations.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

In vivo localization and characterization of functional ciliary neurotrophic factor receptors which utilize JAK-STAT signaling.

The ciliary neurotrophic factor receptor is critically involved in embryonic motor neuron development. Postnatally, it may contribute to neuronal maintenance and regeneration. In addition, pharmacological stimulation of the receptor may slow the progression of several neurodegenerative disorders. The widespread nervous system expression of ciliary neurotrophic factor receptor components and the effects of low ciliary neurotrophic factor concentrations on a wide variety of cells in culture combine to suggest that functional ciliary neurotrophic factor receptors are expressed by many classes of neurons in vivo. However, the in vivo signaling properties and distribution of functional ciliary neurotrophic factor receptors have not been directly determined. We developed a novel in vivo assay of functional ciliary neurotrophic factor receptors which revealed that, in the adult nervous system, cranial and spinal motor neurons are very sensitive to ciliary neurotrophic factor and display a rapid, robust increase in phospho-STAT3 in their dendrites, cell bodies and nuclei, which is specifically blocked by the ciliary neurotrophic factor receptor antagonist, AADH-CNTF. In distinct contrast, several other classes of ciliary neurotrophic factor receptor expressing neurons fail to increase phospho-STAT3 levels following ciliary neurotrophic factor treatment, even when ciliary neurotrophic factor is applied at high concentrations. Leukemia inhibitory factor and epidermal growth factor elicit the same cell-type-dependent pattern of phospho-STAT3 increases. Responsive and non-responsive neurons express comparable levels of STAT3.Therefore, in vivo ciliary neurotrophic factor receptor-initiated STAT3 signal transduction is regulated in a very cell-type-dependent manner. The present data suggest that at least some of this regulation occurs at the STAT3 tyrosine phosphorylation step. These unexpected results also suggest that other forms of receptor-initiated STAT3 signal transduction may be similarly regulated.

Amino Acid Sequence↗
PubMed2000

Leptin receptor-mediated regulation of cholinergic neurotransmitter phenotype in cells of central nervous system origin.

Leptin is an adipocyte-secreted hormone that regulates body weight and exerts effects on hematopoiesis, reproduction, and immunity. The leptin receptor (OBR) shares sequence similarity and signaling capabilities with receptors for cytokines of the ciliary neurotrophic factor (CNTF) family. Our previous finding that CNTF and leptin exert similar anti-obesity effects and activate common neuronal signaling pathways, prompted us to investigate whether leptin may share with CNTF the ability to regulate the expression of specific neuronal genes. To this end, we established a cell line, derived from the murine septal cholinergic neuronal cell line SN-56, which stably expresses OBR. In this cell line, termed SN-56/OBR, leptin induces STAT transcription factor activation and STAT-dependent reporter gene expression in a manner similar to that of CNTF. Furthermore, in SN-56/OBR cells both CNTF and leptin produce changes in neurotransmitter and neuropeptide phenotype characteristic of cholinergic neurons, such as an increase in choline acetyltransferase and vasoactive intestinal polypeptide, and a decrease in neuropeptide Y expression. SN-56/OBR cells thus constitute an interesting new model system to investigate leptin action in cells of central nervous system origin. Possible physiological implications of OBR's intrinsic ability to regulate cholinergic phenotypic markers are discussed.

Alternative Splicing↗
PubMed2000

Enhancing B- and T-cell immune response to a hepatitis C virus E2 DNA vaccine by intramuscular electrical gene transfer.

We describe an improved genetic immunization strategy for eliciting a full spectrum of anti-hepatitis C virus (HCV) envelope 2 (E2) glycoprotein responses in mammals through electrical gene transfer (EGT) of plasmid DNA into muscle fibers. Intramuscular injection of a plasmid encoding a cross-reactive hypervariable region 1 (HVR1) peptide mimic fused at the N terminus of the E2 ectodomain, followed by electrical stimulation treatment in the form of high-frequency, low-voltage electric pulses, induced more than 10-fold-higher expression levels in the transfected mouse tissue. As a result of this substantial increment of in vivo antigen production, the humoral response induced in mice, rats, and rabbits ranged from 10- to 30-fold higher than that induced by conventional naked DNA immunization. Consequently, immune sera from EGT-treated mice displayed a broader cross-reactivity against HVR1 variants from natural isolates than sera from injected animals that were not subjected to electrical stimulation. Cellular response against E2 epitopes specific for helper and cytotoxic T cells was significantly improved by EGT. The EGT-mediated enhancement of humoral and cellular immunity is antigen independent, since comparable increases in antibody response against ciliary neurotrophic factor or in specific anti-human immunodeficiency virus type 1 gag CD8(+) T cells were obtained in rats and mice. Thus, the method described potentially provides a safe, low-cost treatment that may be scaled up to humans and may hold the key for future development of prophylactic or therapeutic vaccines against HCV and other infectious diseases.

Adenovirus E2 Proteins↗
PubMed1999

Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation.

We show that an electric treatment in the form of high-frequency, low-voltage electric pulses can increase more than 100-fold the production and secretion of a recombinant protein from mouse skeletal muscle. Therapeutical erythopoietin (EPO) levels were achieved in mice with a single injection of as little as 1 microgram of plasmid DNA, and the increase in hematocrit after EPO production was stable and long-lasting. Pharmacological regulation through a tetracycline-inducible promoter allowed regulation of serum EPO and hematocrit levels. Tissue damage after stimulation was transient. The method described thus provides a potentially safe and low-cost treatment for serum protein deficiencies.

5' Untranslated Regions↗
PubMed1999

Response of melanocortin-4 receptor-deficient mice to anorectic and orexigenic peptides.

Mutations reducing the functional activity of leptin, the leptin receptor, alpha-melanocyte stimulating hormones (alpha-MSH) and the melanocortin-4 receptor (Mc4r) all lead to obesity in mammals. Moreover, mutant mice that ectopically express either agouti (Ay/a mice) or agouti-related protein (Agrp), antagonists of melanocortin signalling, become obese. These data suggest that alpha-MSH signalling transduced by Mc4r tonically inhibits feeding; however, it is not known to what extent this pathway mediates leptin signalling. We show here that Mc4r-deficient (Mc4r-/-) mice do not respond to the anorectic actions of MTII, an MSH-like agonist, suggesting that alpha-MSH inhibits feeding primarily by activating Mc4r. Obese Mc4r-/-mice do not respond significantly to the inhibitory effects of leptin on feeding, whereas non-obese Mc4r-/- mice do. These data demonstrate that melanocortin signalling transduced by Mc4r is not an exclusive target of leptin action and that factors resulting from obesity contribute to leptin resistance. Leptin resistance of obese Mc4r-/- mice does not prevent their response to the anorectic actions of ciliary neurotrophic factor (CNTF), corticotropin releasing factor (CRF), or urocortin; or the orexigenic actions of neuropeptide Y (NPY) or peptide YY (PYY), indicating that these neuromodulators act independently or downstream of Mc4r signalling.

Animals↗
PubMed1997

Agonistic and antagonistic variants of ciliary neurotrophic factor (CNTF) reveal functional differences between membrane-bound and soluble CNTF alpha-receptor.

Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subunit (CNTFR) and the signal-transducing beta-subunits gp130 and leukemia inhibitory factor receptor-beta (LIFR). CNTFR can function in either membrane-bound or soluble forms. The membrane-bound form mediates the neuronal actions of CNTF, whereas the soluble form serves to confer cytokine responsiveness to non-neuronal cells expressing gp130 and LIFR. The objective of this work was to analyze whether the two receptor isoforms differ in their ability to interact functionally with CNTF and related proteins. Two new types of CNTF variants, characterized by weakened interactions with either CNTFR or both LIFR and gp130, were developed, and the biological activities of these and other mutants were determined in non-neuronal versus neuronal cells, as well as in non-neuronal cells transfected with an expression vector for CNTFR. Membrane anchoring of CNTFR was found to render the CNTF receptor complex relatively insensitive to changes in agonist affinity for either alpha- or beta-receptor subunits and to promote a more efficient interaction with a gp130-depleting antagonistic variant of CNTF. As a result of this phenomenon, which can be rationalized in terms of the multivalent nature of CNTF receptor interaction, CNTF variants display striking changes in receptor selectivity.

Antigens, CD↗
PubMed1997

Ciliary neurotrophic factor corrects obesity and diabetes associated with leptin deficiency and resistance.

Receptor subunits for the neurocytokine ciliary neurotrophic factor (CNTF) share sequence similarity with the receptor for leptin, an adipocyte-derived cytokine involved in body weight homeostasis. We report here that CNTF and leptin activate a similar pattern of STAT factors in neuronal cells, and that mRNAs for CNTF receptor subunits, similarly to the mRNA of leptin receptor, are localized in mouse hypothalamic nuclei involved in the regulation of energy balance. Systemic administration of CNTF or leptin led to rapid induction of the tis-11 primary response gene in the arcuate nucleus, suggesting that both cytokines can signal to hypothalamic satiety centers. Consistent with this idea, CNTF treatment of ob/ob mice, which lack functional leptin, was found to reduce the adiposity, hyperphagia, and hyperinsulinemia associated with leptin deficiency. Unlike leptin, CNTF also reduced obesity-related phenotypes in db/db mice, which lack functional leptin receptor, and in mice with diet-induced obesity, which are partially resistant to the actions of leptin. The identification of a cytokine-mediated anti-obesity mechanism that acts independently of the leptin system may help to develop strategies for the treatment of obesity associated with leptin resistance.

Animals↗
PubMed1996

Identification of ciliary neurotrophic factor (CNTF) residues essential for leukemia inhibitory factor receptor binding and generation of CNTF receptor antagonists.

Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific alpha-receptor subunit (CNTFR alpha) and the signal transducers gp130 and leukemia inhibitory factor receptor-beta (LIFR). The D1 structural motif, located at the beginning of the D-helix of human CNTF, contains two amino acid residues, F152 and K155, which are conserved among all cytokines that signal through LIFR. The functional importance of these residues was assessed by alanine mutagenesis. Substitution of either F152 or K155 with alanine was found to specifically inhibit cytokine interaction with LIFR without affecting binding to CNTFR alpha or gp130. The resulting variants behaved as partial agonists with varying degrees of residual bioactivity in different cell-based assays. Simultaneous alanine substitution of both F152 and K155 totally abolished biological activity. Combining these mutations with amino acid substitutions in the D-helix, which enhance binding affinity for the CNTFR alpha, gave rise to a potent competitive CNTF receptor antagonist. This protein constitutes a new tool for studies of CNTF function in normal physiology and disease.

Amino Acid Sequence↗
PubMed1996

Increased microvascular permeability induced by prolonged interleukin-2 administration is attenuated by the oxygen-free-radical scavenger dimethylthiourea.

Effective use of interleukin (IL)-2 as an antineoplastic agent may be hindered by severe side-effects, in particular vascular leak syndrome, which leads to generalized, especially pulmonary, edema. The oxygen-free-radical scavenger dimethylthiourea (DMTU) was shown to attenuate IL-2-induced vascular leak syndrome in sheep receiving a single IL-2 injection. However, in the clinical setting multiple injections are necessary to gain a therapeutic effect. The present study tests whether DMTU attenuates IL-2-induced vascular leak syndrome following multiple IL-2 injections without affecting IL-2-induced cytotoxicity in peritoneal mononuclear cells. Mice were treated intraperitoneally with 1 x 10(5) units IL-2 three times daily for four consecutive days. DMTU (10 mg/0.5 ml) was administered to the study group once daily, prior to the first IL-2 injection. Comparing the wet/dry weight ratio of lungs, liver, and spleen showed that IL-2 caused a significant (P < 0.05) wet/dry increase in all three organs. DMTU attenuated the wet/dry increase in the lungs (P < 0.05), in the spleen (P < 0.05), and not at all in the liver. IL-2 induced a marked increase in peritoneal mononuclear cell counts, which was not attenuated by DMTU. The cytotoxic effect of IL-2-activated peritoneal mononuclear cells on target B16 cells was also unchanged in animals pretreated with DMTU. In conclusion, we have shown that DMTU ameliorates pulmonary permeability and vascular leak syndrome associated with multiple-dose IL-2 therapy, without eliciting an inhibitory effect on IL-2 induced-cytotoxicity.

Animals↗
PubMed1995

In vitro binding of ciliary neurotrophic factor to its receptors: evidence for the formation of an IL-6-type hexameric complex.

Ciliary neurotrophic factor (CNTF) is a cytokine sharing structural and functional similarities with interleukin-6 (IL-6) and other helical cytokines that utilize the common signalling chain gp130. While IL-6 induces gp130 dimerization, CNTF, after the initial interaction with the specific, non-signalling receptor subunit, CNTFR, induces the formation of gp130/LIF-receptor heterodimers. Through immunoprecipitation experiments with tagged soluble receptor molecules, we recently demonstrated that IL-6 drives the formation of a hexameric receptor complex with a defined topology and composed of two IL-6, two IL-6R alpha and two gp130 molecules. Here, we apply the same strategy to study the assembly in vitro of the CNTF receptor complex. We present evidence that both the cytokine and the specific binding chain undergo dimerization in the presence of gp130. Furthermore, although gp130 and LIFR are able to bind independently to the CNTF/CNTFR sub-complex, they never form homodimers but only heterodimers. We propose that CNTF assembles a hexameric receptor complex composed of two CNTF, two CNTFR, one gp130 and one LIFR molecule, and present a model of the reciprocal interaction of these molecules based on similarities with the IL-6 hexameric complex.

Amino Acid Sequence↗
PubMed1995

CNTF variants with increased biological potency and receptor selectivity define a functional site of receptor interaction.

Human CNTF is a neurocytokine that elicits potent neurotrophic effects by activating a receptor complex composed of the ligand-specific alpha-receptor subunit (CNTFR alpha) and two signal transducing proteins, which together constitute a receptor for leukemia inhibitory factor (LIFR). At high concentrations, CNTF can also activate the LIFR and possibly other cross-reactive cytokine receptors in the absence of CNTFR alpha. To gain a better understanding of its structure-function relationships and to develop analogs with increased receptor specificity, the cytokine was submitted to affinity maturation using phage display technology. Variants with greatly increased CNTFR alpha affinity were selected from a phage-displayed library of CNTF variants carrying random amino acid substitutions in the putative D helix. Selected variants contained substitutions of the wild-type Gln167 residue, either alone or in combination with neighboring mutations. These results provide evidence for an important functional role of the mutagenized region in CNTFR alpha binding. Affinity enhancing mutations conferred to CNTF increased potency to trigger biological effects mediated by CNTFR alpha and enhanced neurotrophic activity on chicken ciliary neurons. In contrast, the same mutations did not potentiate the CNTFR alpha-independent receptor actions of CNTF. These CNTF analogs thus represent receptor-specific superagonists, which should help to elucidate the mechanisms underlying the pleiotropic actions of the neurocytokine.

Amino Acid Sequence↗
PubMed1995

Functional phage display of ciliary neurotrophic factor.

We report the display of human ciliary neurotrophic factor (hCNTF), a survival factor for neuronal cells belonging to the alpha-helical cytokine superfamily, on the surface of the filamentous bacteriophage fd. The hCNTF cDNA was fused to a DNA sequence encoding the C-terminal domain of pIII, a minor coat protein exposed at one end of fd. Gene fusions were cloned into a plasmid containing the ColE1 plasmid and fd origins of replication, and were packaged into phagemid particles upon superinfection with M13KO7 helper phage. The resulting fusion phage bound specifically to anti-CNTF antibodies and to the recombinant soluble CNTF alpha-receptor. Moreover, phage-displayed hCNTF was found to possess biological activity at concentrations comparable to those of the soluble cytokine. These results demonstrate that CNTF can be displayed on phage in a correctly folded and functionally active form. Binding of fusion phage to immobilized CNTF alpha-receptor and subsequent elution at low pH resulted in affinity purification of CNTF-displaying virions. Utilization of this technology should enable the selection of high-affinity variants from libraries of CNTF mutants displayed on phage.

Amino Acid Sequence↗
PubMed1994

Nonradioactive receptor binding assay for ciliary neurotrophic factor.

A nonradioactive receptor binding assay for ciliary neurotrophic factor (CNTF) is described. The assay is based on the interaction between biotinylated human CNTF, soluble gp130, and soluble myc-tagged CNTF receptor captured on a microtiter plate via an antibody against the myc epitope tag. Bound cytokine is revealed by alkaline phosphatase-conjugated avidin. Purified human and rat CNTF competed with biotinylated CNTF for receptor binding, with IC50 values of 29 and 2 nM, respectively. Since the higher affinity of rat vs human CNTF has been previously shown to be conferred by the arginine residue at position 63 of the rat protein, we also tested a human CNTF mutant carrying a Q63R substitution. Secreted forms of wild-type and mutant CNTF were expressed in Escherichia coli, and the amount of cytokines in periplasmic extracts was determined by quantitative Western blotting analysis. The human CNTF mutant (Q63R, N137S) was found to compete with biotinylated CNTF for binding to soluble CNTF receptor with an eightfold higher apparent affinity than wild-type human CNTF. The present method thus faithfully reproduces the relative activities of CNTF analogs determined in other assay systems. The possibility of assaying cytokines in crude bacterial extracts makes the new technique particularly suitable for rapidly determining the receptor binding potencies of genetically engineered CNTF variants.

Amino Acid Sequence↗
PubMed1993

Biotin binders selected from a random peptide library expressed on phage.

Recombinant biotin-binding phages were affinity-selected from a random peptide library expressed on the surface of filamentous phage. Phage binding to biotinylated proteins was half-maximally inhibited by micromolar concentrations of a monobiotinylated molecule. Sequencing of the peptide inserts of selected phages led to the identification of a previously unknown biotin-binding motif, CXWXPPF(K or R)XXC. A synthetic peptide containing this sequence motif inhibited streptavidin binding to biotinylated BSA with an IC50 of 50 microM. This compound represents the shortest non-avidin biotin-binding peptide identified to date. Our results illustrate that phage display technology can be used to identify novel ligands for a small non-proteinaceous molecule.

Amino Acid Sequence↗