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 ob͞ob 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 db͞db 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.

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.Hepatitis C virus (HCV) is the major etiological agent of both community and posttransfusionally acquired non-A, non-B viral hepatitis. Approximately 70% of patients develop chronic hepatitis, of which 20 to 30% progress onto liver cirrhosis, and all cases of infection carry an increased risk of hepatocellular carcinoma (1). Presently, the only available therapies are alpha interferon (IFN-␣) alone or in combination with ribavirin (17,34,45). Such treatments are expensive, show low-response rates, and carry the risk of significant side effects. Consequently, the development of a vaccine against hepatitis C remains a high priority goal.The putative envelope protein E2 of HCV and, in particular, the hypervariable region 1 (HVR1) are the most variable antigenic fragments in the whole viral genome and are the target of neutralizing antibodies (7,16,41). Antibodies against a single E2 HVR1 are isolate specific and lead to the emergence of escape mutants during chronic infection (16,26,27,52,58,63). Thus, the major task in developing a HCV vaccine would be to generate an immunogen that induces a highly crossreactive anti-HVR1 response to prevent the outgrowth of escape mutants rather than require the immune system to deal with them after they arise.A few reports support the notion that cytotoxic-T-lymphocyte (CTL) immunity plays an essential role in limiting HCV infection in humans (38,48,51). Similarly, an earl...

Ciliary neurotrophic factor (CNTF) drives the sequential assembly of a receptor complex containing the ligand-specific ai-receptor subunit (CNTFRei) and the signal transducers gpl3O and leukemia inhibitory factor receptor-a (LIFR). The Dl 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 CNTFRa or gpl3O. 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 CNTFRa, 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.Ciliary neurotrophic factor (CNTF) is a protein expressed by Schwann cells and astrocytes, which exerts potent stimulatory effects on the survival and differentiation of a variety of neuronal and glial cells and has been proposed to act as a lesion factor involved in the prevention of neuronal degeneration following injury (1, 2). Studies of the physiological or pathophysiological roles of CNTF would be facilitated by the availability of specific, high-affinity receptor antagonists. Like other growth factors and cytokines controlling essential cell functions such as survival, proliferation, and differentiation, CNTF exerts its actions through the binding, sequential assembly, and activation of a multi-subunit receptor complex (3, 4). The identification of the protein domains that participate in interactions with different receptor subunits is a prerequisite for understanding the mechanism of receptor activation and for the design of specific antagonists (5-8).CNTF belongs to a group of functionally related proteins of the long-chain a-helical cytokine superfamily, including interleukin-6 (IL-6), leukemia inhibitory factor (LIF), oncostatin M, IL-11, and cardiotrophin-1 (CT-1) (4, 9-12), which signal through structurally related and partially shared receptor subunits. The CNTF receptor complex is composed of a low-affinity, ligand-specific a-receptor (CNTFRa), which is predominantly expressed in neuronal cells (13), and two more widely distributed signaltransducing subunits, namely gp13O and the LIF receptor-13 (LIFR). Binding of CNTF to CNTFRa triggers the subsequent association ofgp13O and LIFR in a high-affinity receptor complex (3). By analogy to the hexameric receptor complex assembled by , the CNTF receptor is thought to contain two cytokine molecules, two a-receptor and two signal-transducing subunits (15). Heterodimerization of the latter leads to the a...

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The application of a phosphoramidate prodrug approach to 2'-C-methylcytidine (NM107), the first nucleoside inhibitor of the hepatitis C virus (HCV) NS5B polymerase, is reported. 2'-C-Methylcytidine, as its valyl ester prodrug (NM283), was efficacious in reducing the viral load in patients infected with HCV. Several of the phosphoramidates prepared demonstrated a 10- to 200-fold superior potency with respect to the parent nucleoside in the cell-based replicon assay. This is due to higher levels of 2'-C-methylcytidine triphosphate in the cells. These prodrugs are efficiently activated and converted to the triphosphate in hepatocytes of several species. Our SAR studies ultimately led to compounds that gave high levels of NTP in hamster and rat liver after subcutaneous dosing and that were devoid of the toxic phenol moiety usually found in ProTides.