Antitumor activity of recombinant adenoviral vectors expressing murine IFN-alpha in mice injected with metastatic IFN-resistant tumor cells.

Recent studies have shown that gene therapy with type I interferon (IFN) in an adenovirus vector is a powerful tool to suppress the growth of human tumors transplanted in immune-deficient mice. However, in these studies the host immune-mediated effects, which may be important in mediating the long-term control of tumor growth by these cytokines, was not studied. In this paper, we evaluate the antitumor efficacy of different adenoviral vectors containing mouse IFN-alpha genes (i.e., a first-generation replication-defective vector containing IFN-alpha1 and two different second-generation vectors containing IFN-alpha2) in immunocompetent DBA/2 mice transplanted with highly metastatic Friend leukemic cells resistant in vitro to type I IFN. We found that injection of all the different adenovirus vectors containing mouse IFN-alpha( genes resulted in a marked antitumor response in mice transplanted either subcutaneously or intravenously with IFN-resistant Friend leukemic cells compared to tumor-bearing animals inoculated with a control vector. Tumor growth inhibition after injection of IFN-adenovirus vectors was associated with a prolonged presence of high IFN levels in the sera of the injected mice. Suppression of metastatic tumor growth was also observed after a single injection of the IFN--adenovirus recombinant vectors, whereas a comparable antitumor response generally required several injections of high doses of IFN. Altogether, these results demonstrate that IFN--adenoviral vectors can efficiently inhibit metastatic tumor growth by host-mediated mechanisms and suggest that adenovirus-mediated IFN-alpha gene therapy may represent an attractive alternative to the conventional clinical use of this cytokine, which generally requires multiple injections of high IFN doses for a prolonged period of time.

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.

Liver-specific alpha 2 interferon gene expression results in protection from induced hepatitis.

The current therapy for hepatitis B and C is based on systemic administration of recombinant human alpha interferon (r-hIFN-alpha). However, systemic delivery of r-hIFN-alpha is associated with severe side effects, but more importantly, it is effective in only a small percentage of patients. In an effort to maximize IFN-alpha antiviral efficacy, we have explored the therapeutic potential of murine IFN-alpha2 (mIFNalpha2) selectively expressed in the liver. To this end, we have developed a helper-dependent adenovirus vector (HD) containing the mIFN-alpha2 gene under the control of the liver-specific transthyretin promoter (HD-IFN). Comparison with a first-generation adenovirus carrying the same mIFN-alpha2 expression cassette indicates that at certain HD-IFN doses, induction of antiviral genes can be achieved in the absence of detectable circulating mIFN-alpha2. Challenge of injected mice with mouse hepatitis virus type 3 showed that HD-IFN provides high liver protection. Moreover, liver protection was also observed in acute nonviral liver inflammation hepatitis induced by concanavalin A at 1 month postinfection. These results hold promise for the development of a gene therapy treatment for chronic viral hepatitis based on liver-restricted expression of IFN-alpha2.

Prolonged expression and effective readministration of erythropoietin delivered with a fully deleted adenoviral vector.

Helper-dependent (HD) adenoviral (Ad) vectors, in which all viral coding sequences are deleted, have been generated. We show here that intravenous delivery of a mouse EPO (mEPO) expression cassette cloned in an HD vector in immunocompetent mice is effective and long lasting, but not permanent. A precise dose-response relationship between the dose of injected virus and stable EPO serum levels was observed, together with a 100-fold increase in gene expression per infectious particle when compared with a first-generation Ad vector bearing the same cassette. As a direct consequence, therapeutic increases in hematocrit that lasted more than 6 months were achieved with minute amounts of virus, which caused no detectable production of neutralizing antibodies. Intravenous readministration of the HD-mEPO vector in the same mice was as effective as in naive animals without any need for prior immunosuppression. Finally, HD-mEPO injection in subtotally nephrectomized rats improved the anemic status induced by surgery. HD Ad vectors are thus excellent tools for EPO gene therapy.

Selection of functional variants of the NS3-NS4A protease of hepatitis C virus by using chimeric sindbis viruses.

The NS3-NS4A serine protease of hepatitis C virus (HCV) mediates four specific cleavages of the viral polyprotein and its activity is considered essential for the biogenesis of the HCV replication machinery. Despite extensive biochemical and structural characterization, the analysis of natural variants of this enzyme has been limited by the lack of an efficient replication system for HCV in cultured cells. We have recently described the generation of chimeric HCV-Sindbis viruses whose propagation depends on the NS3-NS4A catalytic activity. NS3-NS4A gene sequences were fused to the gene coding for the Sindbis virus structural polyprotein in such a way that processing of the chimeric polyprotein, nucleocapsid assembly, and production of infectious viruses required NS3-NS4A-mediated proteolysis (G. Filocamo, L. Pacini, and G. Migliaccio, J. Virol. 71:1417-1427, 1997). Here we report the use of these chimeric viruses to select and characterize active variants of the NS3-NS4A protease. Our original chimeric viruses displayed a temperature-sensitive phenotype and formed lysis plaques much smaller than those formed by wild-type (wt) Sindbis virus. By serially passaging these chimeric viruses on BHK cells, we have selected virus variants which formed lysis plaques larger than those produced by their progenitors and produced NS3-NS4A proteins different in size and/or sequence from those of the original viruses. Characterization of the selected protease variants revealed that all of the mutated proteases still efficiently processed the chimeric polyprotein in infected cells and also cleaved an HCV substrate in vitro. One of the selected proteases was expressed in a bacterial system and showed a catalytic efficiency comparable to that of the wt recombinant protease.

Lipofection of purified adeno-associated virus Rep68 protein: toward a chromosome-targeting nonviral particle.

Adeno-associated virus (AAV) integrates very efficiently into a specific site (AAVS1) of human chromosome 19. Two elements of the AAV genome are sufficient: the inverted terminal repeats (ITRs) and the Rep78 or Rep68 protein. The incorporation of the AAV integration machinery in nonviral delivery systems is of great interest for gene therapy. We demonstrate that purified recombinant Rep68 protein is functionally active when directly delivered into human cells by using the polycationic liposome Lipofectamine, promoting the rescue-replication of a codelivered ITR-flanked cassette in adenovirus-infected cells and its site-specific integration in noninfected cells. The sequencing of cloned virus-host DNA junctions confirmed that lipofected Rep68 protein triggers site-specific integration at the same sites in chromosome 19 already characterized in cells latently infected with AAV.

Mismatch repair deficiency associated with overexpression of the MSH3 gene.

We tested the ability of recombinant hMutSalpha (hMSH2/hMSH6) and hMutSbeta (hMSH2/hMSH3) heterodimers to complement the mismatch repair defect of HEC59, a human cancer cell line whose extracts lack all three MutS homologues. Although repair of both base/base mispairs and insertion-deletion loops was restored by hMutSalpha, only the latter substrates were addressed in extracts supplemented with hMutSbeta. hMutSalpha was also able to complement a defect in the repair of base/base mispairs in CHO R and HL60R cell extracts. In these cells, methotrexate-induced amplification of the dihydrofolate reductase (DHFR) locus, which also contains the MSH3 gene, led to an overexpression of MSH3 and thus to a dramatic change in the relative levels of MutSalpha and MutSbeta. As a rule, MSH2 is primarily complexed with MSH6. MutSalpha is thus relatively abundant in mammalian cell extracts, whereas MutSbeta levels are generally low. In contrast, in cells that overexpress MSH3, the available MSH2 protein is sequestered predominantly into MutSbeta. This leads to degradation of the partnerless MSH6 and depletion of MutSalpha. CHO R and HL60R cells therefore lack correction of base/base mispairs, whereas loop repair is maintained by MutSbeta. Consequently, frameshift mutations in CHO R are rare, whereas transitions and transversions are acquired at a rate two orders of magnitude above background. Our data thus support and extend the findings of Drummond et al. [Drummond, J. T., Genschel, J., Wolf, E. & Modrich, P. (1997) Proc. Natl. Acad. Sci. USA 94, 10144-10149] and demonstrate that mismatch repair deficiency can arise not only through mutation or transcriptional silencing of a mismatch repair gene, but also as a result of imbalance in the relative amounts of the MSH3 and MSH6 proteins.

Rational design and functional expression of a constitutively active single-chain NS4A-NS3 proteinase.

BACKGROUND: The proteinase domain of the hepatitis C virus NS3 protein is involved in the maturation of the viral polyprotein. A central hydrophobic domain of the NS4A protein is required as a cofactor for its proteolytic activity. The three-dimensional structure of the proteinase domain alone and complexed with an NS4A-derived peptide has been solved recently and revealed that the N terminus of the proteinase is in near proximity to the C terminus of the cofactor. To study the molecular basis of the enzyme activation by its cofactor and to overcome the difficulties of structural and functional investigation associated with a two-species complex, we rationally designed a link to bridge the two molecules in order to have a single polypeptide construct. RESULTS: The engineered construct led to the production of a stable, monomeric protein with proteolytic activity that is independent from the addition of a synthetic peptide representing the cofactor domain of the NS4A protein. The protein is active on both protein and synthetic peptide substrates. Spectroscopic and kinetic analysis of the recombinant NS4A-NS3 single-chain proteinase demonstrated features superimposable with the isolated NS3 proteinase domain complexed with the NS4A cofactor. CONCLUSIONS: We designed a very tight connection between the NS3 and NS4A polypeptide chains with the rationale that this would allow a more stable structure to be formed. The engineered single-chain enzyme was indistinguishable from the NS3 proteinase complexed with its NS4A cofactor in all enzymatic and physico-chemical properties investigated.

Immunogenicity of filamentous phage displaying peptide mimotopes after oral administration.

Selected human sera can be used to identify disease-related peptide epitopes (mimotopes) displayed on bacteriophages. Parenteral administration of such recombinant phages is an effective route of immunization in different experimental animals, indicating that mimotopes could be an important source of leads for new vaccines. Here it is shown that intranasal or intragastric administration of phage in mice induces an immunological response both to the wild type proteins of the phage and to mimotopes displayed on them. Using mimotopes of human HBV surface antigen and of human HCV peptides, the authors show that the response induced by oral administration is specifically cross-reactive with the original antigen. These findings indicate that phage displaying selected mimotopes could be useful for the development of orally effective vaccines.

Shc mediates IL-6 signaling by interacting with gp130 and Jak2 kinase.

IL-6 is a multifunctional cytokine involved in hemopoiesis, immune regulation, inflammation, neural development, and infection. IL-6 belongs to a family of related cytokines that includes leukemia inhibitory factor, oncostatin M, IL-11, ciliary neurotropic factor, and cardiotropin-1, all of which initiate signaling through a receptor-associated gp130. IL-6 induces homodimerization of gp130 and activates the Jak/STAT pathway of signal transduction. In addition, IL-6 stimulates the mitogen-activated protein kinases designated ERK (extracellular signal-regulated kinase)-1 and -2. Activation of ERK-1 and -2 may involve the Src homology-2 containing proteins Shc and Grb2. Here we provide evidence that Shc could function as signaling molecules for IL-6 in DeFew-IL-6R/gp130 cells, a human B lymphoma cell line engineered to express high levels of both the IL-6R (p80) and the gp130 subunit. IL-6 was shown to promote the rapid tyrosine phosphorylation of gp130, Jak2, and Shc proteins. Moreover, Shc associated both in vivo and in vitro with phosphorylated gp130 through the Shc-Src homology-2 domain. We also report that Shc bound to activated Jak2 by using the Shc amino terminal phosphotyrosine interaction domain. Following IL-6 stimulation, Shc physically associated with Grb2. Thus, the data point to Shc proteins as a functional link between the Jak2 and Ras pathways of IL-6 signal transduction.

Selection of antigenic and immunogenic mimics of hepatitis C virus using sera from patients.

Using sera from hepatitis C virus (HCV)-infected patients and noninfected subjects to screen random peptide libraries displayed on phage, we selected peptides specifically reacting with sera from infected patients. These phage- borne peptides were shown to mimic distinct HCV determinants. They detected in all cases the presence of anti-HCV Abs in a large panel of patients' sera, thus demonstrating the high sensitivity of the selected peptides as diagnostic markers. In addition, this diagnostic approach allowed a detailed characterization of the individual humoral response to viral infection. Phage-displayed HCV mimics were substitutes for the authentic HCV epitopes in inducing a strong specific response against HCV when used as immunogens in mice. These results support the search for HCV mimics with the potential to elicit a protective immune response as leads for the development of a mimotope-based vaccine against viral infection.

Derivation of vaccines from mimotopes. Immunologic properties of human hepatitis B virus surface antigen mimotopes displayed on filamentous phage.

We have previously reported the identification, using human immune sera, of mimotopes of human hepatitis B virus surface Ag (HBsAg) displayed on filamentous phage. To test if these mimotopes could be useful in developing a vaccine against the human hepatitis B virus (HBV), we have compared the humoral immune response of animals immunized either with a recombinant HBsAg vaccine, or with mimotopes. Immunogens were prepared by fusing the mimotopes on different carrier molecules (phage coat protein pIII and pVIII, recombinant human H ferritin, HBV core peptide) and by synthesizing multiple antigenic peptides carrying the mimotopes' amino acid sequences. These immunogens were injected into mice and rabbits and sera were collected and tested for the presence of HBsAg-specific Abs. Our data confirm that mimotopes can induce a humoral immune response resembling that induced by the original Ag, and HBsAg mimotopes displayed on phage prove to be the best immunogens, inducing the most reproducible and potent immunization. Mimotopes that react as HBV subtype-specific Ags do not show this specificity as immunogen and induce a nonsubtype-restricted response. Furthermore, mimotopes displayed on phage elicit a strong response to HBsAg in a strain of mouse reported to show a low response to it. These results indicate that mimotopes identified from random peptide libraries through utilizing human immune sera could be important leads for the derivation of new vaccines.

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.

Monoclonal antibodies that recognise filamentous phage: tools for phage display technology.

We generated six hybridoma cell lines that secrete monoclonal antibodies (mAb) which specifically bind filamentous phage coat proteins. Two of these mAb recognise epitopes that include the N terminus of the coat protein III (pIII), while two others are specific for the N terminus of the major coat protein VIII (pVIII). These mAb are valuable tools to study phage assembly and structure. Furthermore, we describe two examples of how these mAb can be exploited in the construction and screening of peptide libraries displayed by the filamentous phase major coat protein. We have used one of these mAb to develop a sensitive ELISA with crude phage supernatants. This assay allows rapid screening of large numbers of clones from random peptide phage libraries. Some of the anti-phage mAb described here can interfere with wild-type phage propagation, while phage carrying modifications in their coat proteins are insensitive to growth inhibition. We have exploited this observation as a tool to favour the growth of phage displaying peptides fused to pVIII, with respect to vector phage.

Oncostatin M binds directly to gp130 and behaves as interleukin-6 antagonist on a cell line expressing gp130 but lacking functional oncostatin M receptors.

Oncostatin M (OM) and interleukin 6 (IL-6) are functionally related cytokines, which trigger similar biological responses because they share gp130 as a common signal transducing transmembrane receptor. While IL-6 recruits gp130 only upon binding to its specific receptor subunit (IL-6R alpha), reconstitution and cross-linking experiments on cell membranes suggest that OM can directly interact with gp130 and that this interaction is necessary but not sufficient to stimulate cells. However, the issue of the direct binding between gp130 and OM, in the absence of any additional membrane component, remained essentially unclarified. In this paper we show that, uniquely among the family of cytokines that transduce through gp130, OM directly binds in vitro with a 10(-8) M affinity sgp130, a soluble form of gp130. Moreover, titration of sgp130 with OM inhibits the formation of a ternary complex comprising IL-6, sIL-6R alpha, and sgp130. These in vitro properties of OM are consistent with the additional finding that on human hepatoma Hep3B cells, which express gp130 but not functional OM receptors, OM does not mimic IL-6 activity, but rather behaves, at high doses, as an IL-6 antagonist.

A Multimeric Synthetic Peptide Combinatorial Library.

We describe here a novel type of synthetic peptide library, named Multimeric Synthetic Peptide Combinatorial Library (M-SPCL), where multiple small peptide ligands are tied together in the same molecule. The advantage of using small peptides in the form of M-SPCL is two-fold: first, the high density assembly of the sequences on the branching scaffold leads to signal amplification, thereby effectively lowering the binding threshold for the selection of ligands; second, to interfere with protein-protein interactions, multimericity has been shown to be a desirable feature per se. The M-SPCL is prepared by solid-phase peptide synthesis, based on the structure of Multiple Antigen Peptides. When prepared in Positional Scanning format [C. Pinilla, J. Appel, P. Blanc and R.A. Houghten. 1992. BioTechniques 13: 901-905], selection is based on the amplified interaction of a single residue in a sequence-defined position. The usefulness of the new library was demonstrated by the selection of octameric peptides, which inhibit the binding of the cytokine human interleukin-6 to its receptor, with an apparent nanomolar affinity. Tetrameric, but not dimeric, branched peptides with the same sequences were also active with comparable affinity. The success of this approach is noteworthy, since screening of the corresponding monomeric pentapeptide SPCL did not lead to the selection of any inhibitory compound in the same system.

[AIDS and pedodontics: the real risk and its prevention]. / AIDS e pedodonzia: rischio reale e sua prevenzione.

The daily increase in carriers of the AIDS virus (150,000 is the latest estimate for Italy) means that the dentist must pay the utmost attention in selecting cases at risk and in defending himself from the possibility of contagion. Albeit to a lesser extent, the pedodontist is also involved in this problem. Here situations in which the risk of contagion is greatest because of the patient's social position or associated pathologies and the difficulty of the operation required are reported and some indications are offered to guide the pedodontist's professional behaviour.

[A case of Ehlers-Danlos syndrome]. / Su un caso di sindrome di Ehlers-Danlos (E.D.)

A case of Ehlers-Danlos syndrome, personally observed, is described. Stress is laid on the rarity of this syndrome (less than 200 cases in the world literature) and on its peculiar symptoms. The familial character of this syndrome was not proved in the case observed.