[Show abstract][Hide abstract] ABSTRACT: IFN-α/β link innate and adaptive immune responses by directly acting on naïve CD8(+) T cells. This concept unveiled in mice remains unexplored in humans. To investigate that, human CD8(+) CD45RO(-) cells were stimulated with beads coated with anti-CD3 and anti-CD28 mAb, mimicking Ag (type-1) and co-stimulatory (type-2) signals, in the presence or absence of IFN-α and their transcriptional profiles were defined by cDNA-microarrays. We show that IFN-α provides a strong third signal directly to human CD8(+) T cells resulting in regulation of critical genes for their overall activation. This transcriptional effect was substantiated at the protein level and verified by functional assays. Interestingly, the biological effects derived from this stimulation vary depending on the CD8(+) T-cell population. Thus, whereas IFN-α increases the proliferative capacity of naïve CD8(+) T cells, it inhibits or does not affect the proliferation of Ag-experienced cells, such as memory and effector CTL, including CMV-specific lymphocytes. Cytolysis and IFN-γ-secretion of all these populations are enhanced by IFN-α-derived signals, which are critical in naïve CD8(+) T cells for acquisition of effector functions. Our findings in human CD8(+) T cells are informative to understand and improve IFN-α-based therapies for viral and malignant diseases.
European Journal of Immunology 12/2010; 40(12):3389-402. DOI:10.1002/eji.201040664 · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic hepatitis B is a major cause of liver-related death worldwide. Interleukin-12 (IL-12) induction accompanies viral clearance in chronic hepatitis B virus infection. Here, we tested the therapeutic potential of IL-12 gene therapy in woodchucks chronically infected with woodchuck hepatitis virus (WHV), an infection that closely resembles chronic hepatitis B. The woodchucks were treated by intrahepatic injection of a helper-dependent adenoviral vector encoding IL-12 under the control of a liver-specific RU486-responsive promoter. All woodchucks with viral loads below 10(10) viral genomes (vg)/ml showed a marked and sustained reduction of viremia that was accompanied by a reduction in hepatic WHV DNA, a loss of e antigen and surface antigen, and improved liver histology. In contrast, none of the woodchucks with higher viremia levels responded to therapy. The antiviral effect was associated with the induction of T-cell immunity against viral antigens and a reduction of hepatic expression of Foxp3 in the responsive animals. Studies were performed in vitro to elucidate the resistance to therapy in highly viremic woodchucks. These studies showed that lymphocytes from healthy woodchucks or from animals with low viremia levels produced gamma interferon (IFN-gamma) upon IL-12 stimulation, while lymphocytes from woodchucks with high viremia failed to upregulate IFN-gamma in response to IL-12. In conclusion, IL-12-based gene therapy is an efficient approach to treat chronic hepadnavirus infection in woodchucks with viral loads below 10(10) vg/ml. Interestingly, this therapy is able to break immunological tolerance to viral antigens in chronic WHV carriers.
Journal of Virology 03/2009; 83(6):2663-74. DOI:10.1128/JVI.02384-08 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interferon alpha (IFNalpha) is the first line treatment for chronic hepatitis B and C. In order to test new IFNalpha delivery systems and investigate the function of this cytokine in the woodchuck model, the best animal model of chronic hepatitis B, we produced and purified recombinant woodchuck IFNalpha and used it to produce monoclonal antibodies. wIFNalpha5 was cloned in a prokaryotic expression system, expressed as His-tagged protein and then purified. The rwIFNalpha5 protein was found to induce STAT-3 phosphorylation, to enhance 2',5'-oligoadenylate synthetase mRNA levels and to possess a potent antiviral activity. Two monoclonal antibodies were obtained through immunization of rats with rwIFNalpha5. Both recognized rwIFNalpha5 in western blot analysis and one was able to neutralize the antiviral activity of the rwIFNalpha5 and lymphoblastoid IFNalpha preparations. Finally, a capture rwIFNalpha5 ELISA was developed using both antibodies. In summary, the tools generated in this study will allow different forms of IFNalpha delivery as well as different combination therapies in woodchuck hepatitis virus infection to be tested, thus providing useful information for the design of new strategies to treat chronic hepatitis B in humans.
Journal of interferon & cytokine research: the official journal of the International Society for Interferon and Cytokine Research 12/2008; 29(2):75-82. DOI:10.1089/jir.2008.0012 · 2.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An adeno-associated virus (AAV) derived vector in gene transfer model that induces IGF-1 expression could repair articular cartilage.
Male Wistar rats, 150 and 200 g, and 7 weeks old, were used. Effectiveness of constructed vectors was assayed inoculating them in rat knees of control and damaged animals either mechanically or by collagen-induced arthritis. Inoculation was intra-articular with 50 microL of recombinant AAV-Luciferase (1.25 x 10(8) particles). The rats were killed after 1, 2, 4 and 8 weeks. IGF-I activity was analyzed by injecting 50 microL of recombinant AAV (1.25 x 10(8) particles) in animals with damaged knees. Final analysis was performed after 8 weeks.
The activity of AAV vectors in vitro shows the presence of mRNA coding to IGF-I in cells infected with AAV-IGF and not in control cells without viral vectors and an increase in secreted IGF-I protein in culture medium. In vivo, AAV derived vectors induced protein expression in cartilage 2 months after inoculation. In the animals killed after 1 and 2 weeks, no significant increase in the reaction of luciferase was observed (P > 0.05). In the group of animals with no injury an increase was observed at 4 weeks, which was more marked and significant after 8 weeks (P = 0.029). The same behavior occurred in the animals with induced arthritis and in the mechanical injury group. In the levels of expression after 8 weeks, no significant differences were found between the two groups of injured animals and the group of healthy animals infected with the virus. The joints of the animals that were subjected to injuries in the cartilage and inoculated with AAV-IGF-I presented a similar appearance to those animals inoculated with saline solution.
Autoimmune and mechanical lesions did not show improvement in the state of its cartilage after the treatment. The use of AAV vectors capable of inducing the expression of IGF-I in vitro is therefore not sufficient to protect the cartilage from the serious damage.
Archives of Orthopaedic and Trauma Surgery 02/2008; 128(2):239-47. DOI:10.1007/s00402-007-0407-7 · 1.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Woodchucks infected with the woodchuck hepatitis virus (WHV) is the best available animal model for testing the immunotherapeutic effects of dendritic cells (DCs) in the setting of a chronic infection, as woodchucks develop a persistent infection resembling that seen in humans infected with the hepatitis B virus. In the present study, DCs were generated from woodchuck peripheral blood mononuclear cells (wDCs) in the presence of human granulocyte macrophage colony-stimulating factor (hGM-CSF) and human interleukin 4 (hIL-4). After 7 days of culture, cells with morphology similar to DCs were stained positively with a cross-reactive anti-human CD86 antibody. Functional analysis showed that uptake of FITC-dextran by wDCs was very efficient and was partially inhibited after LPS-induced maturation. Furthermore, wDCs stimulated allogenic lymphocytes and induced proliferation. Moreover, wDCs were transduced efficiently with a human adenovirus serotype 5 for the expression of beta-galactosidase. Following transduction and in vivo administration of such DCs into woodchucks, an antigen-specific cellular immune response was induced. These results demonstrate that wDCs can be generated from the peripheral blood. Following transfection with a recombinant adenovirus wDCs can be used as a feasible and effective tool for eliciting WHV-specific T-cell responses indicating their potential to serve as prophylactic and therapeutic vaccines.
Journal of Medical Virology 05/2007; 79(5):522-9. DOI:10.1002/jmv.20808 · 2.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: The access of viral particles to the bloodstream and organic fluids after intratumoral injection of adenoviral vectors for cancer gene therapy may influence their toxicity profile and may result in an environmental risk.Material and Methods: Patients under study were enrolled in two phase I clinical trials evaluating the feasibility and safety of intratumoral injection of an adenoviral vector encoding either human interleukin-12 genes (AdIL-12) or viral thymidine kinase (AdTK). Twenty-one patients with pancreatic (7), colorectal (5), or primary liver (9) malignancies received Ad.IL-12 in doses ranging from 2.5 × 10e10 to 3 × 10e12 viral particles. Eleven patients with pancreatic (4) or hepatocellular (7) carcinomas received Ad-TK in doses ranging from 2 × 10e10 to 2 × 10e11 viral particles. PFU to viral particle ratio were 12.65 and 130 for Ad.IL-12 and AdTK, respectively. In both trials the virus was administered by direct intratumoral injection either percutaneously (into liver tumor nodules) or endoscopically (into primary pancreatic tumors). Serum samples were taken immediately before and 5, 15, 60 and 120 minutes after the end of each intratumoral injection and then 1, 2, 3, 4, 5, 7, 10, 13 and 17 days after. Saliva, sputum, feces, and urine samples were taken before and 1, 2, 3, 4 and 5 days after adenoviral injection. Viral DNA was extracted and real quantitative PCR was performed. The infectivity of the viral particles eventually found in saliva, urine and feces was analyzed in vitro by infection of 293 cellsResults: We have found a direct correlation between the dose of virus injected and the intensity and duration of the adenoviral load in serum. An increase in the titers of total and neutralizing antiadenoviral antibodies was also universally observed, although significant differences were observed depending on the age of the patients. Neutralizing antibody titers inversely correlate with age. In those patients that were retreated, a higher serum viral load was observed after the first administration than after second or third injections, probably reflecting sequestration mediated by neutralizing antibodies. Viral particles were detected in some of these patients at day 1 and sporadically day 2, in saliva (12), urine (2) and feces (3). Interestingly, patients that had viral particles in feces were those that had their primary pancreatic tumors injected by endoscopic- ultrasound guidance. Most importantly, none of the samples of saliva, urine and feces that were positive for viral particles showed infectious viral particles (while incubation of the same samples with control virus resulted in only a partial inhibition of its infectivity).In conclusion, our data show that viral shedding largely depends on the dose administered and the route of administration. And they also strongly suggest that no especial containment measures are required for patients receiving intratumoral injection of defective adenovirus.
[Show abstract][Hide abstract] ABSTRACT: Transforming growth factor-beta (TGF-beta) plays a crucial role in the pathogenesis of skin fibrotic diseases. Systemic TGF-beta inhibitors effectively inhibit fibrosis in different animal models; however, systemic inhibition of TGF-beta raises important safety issues because of the pleiotropic physiological effects of this factor. In this study, we have investigated whether topical application of P144 (a peptide inhibitor of TGF-beta1) ameliorates skin fibrosis in a well-characterized model of human scleroderma. C3H mice received daily subcutaneous injections of bleomycin for 4 wk, and were treated daily with either a lipogel containing P144 or control vehicle. Topical application of P144 significantly reduced skin fibrosis and soluble collagen content. Most importantly, in mice with established fibrosis, topical treatment with P144 lipogel for 2 wk significantly decreased skin fibrosis and soluble collagen content. Immunohistochemical studies in P144-treated mice revealed a remarkable suppression of connective tissue growth factor expression, fibroblast SMAD2/3 phosphorylation, and alpha-smooth muscle actin positive myofibroblast development, whereas mast cell and mononuclear cell infiltration was not modified. These data suggest that topical application of P144, a peptide inhibitor of TGF-beta1, is a feasible strategy to treat pathological skin scarring and skin fibrotic diseases for which there is no specific therapy.
[Show abstract][Hide abstract] ABSTRACT: Preventive and therapeutic vaccine strategies aimed at controlling hepatitis C virus (HCV) infection should mimic the immune responses observed in patients who control or clear HCV, specifically T helper (Th) type 1 and CD8+ cell responses to multiple antigens, including nonstructural protein (NS) 3. Given the experience with human immunodeficiency virus, the best candidates for this are based on DNA prime, pox, or adenovirus boost regimens.
In rhesus macaques, we compared NS3-expressing DNA prime and adenovirus boost strategy with 2 alternative priming approaches aimed at modifying Th1 and CD8+ responses: DNA adjuvanted with interleukin (IL)-2- and -12-encoding plasmids or Semliki Forest virus (SFV).
All prime-boost regimens elicited NS3-specific B and T cell responses in rhesus macaques, including CD8+ responses. SFV priming induced higher lymphoproliferation and longer Th1 memory responses. The use of IL-2- and IL-12-expressing vectors resulted in reduced Th2 and antibody responses, which led to increased Th1 skewing but not to an increase in the magnitude of the IFN- gamma and CD8+ responses.
All strategies induced Th1 cellular responses to HCV NS3, with fine modulations depending on the different priming approaches. When they are developed for more HCV antigens, these strategies could be beneficial in therapeutic vaccine approaches.
The Journal of Infectious Diseases 10/2005; 192(5):920-9. DOI:10.1086/432517 · 6.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the course of a clinical trial consisting of intratumoral injections of dendritic cells (DCs) transfected to produce interleukin-12, the use of (111)In-labeled tracing doses of DCs showed that most DCs remained inside tumor tissue, instead of migrating out. In search for factors that could explain this retention, it was found that tumors from patients suffering hepatocellular carcinoma, colorectal or pancreatic cancer were producing IL-8 and that this chemokine attracted monocyte-derived dendritic cells that uniformly express both IL-8 receptors CXCR1 and CXCR2. Accordingly, neutralizing antihuman IL-8 monoclonal antibodies blocked the chemotactic attraction of DCs by recombinant IL-8, as well as by the serum of the patients or culture supernatants of human colorectal carcinomas. In addition, tissue culture supernatants of colon carcinoma cells inhibited DC migration induced by MIP-3beta in an IL-8-dependent fashion. IL-8 production in malignant tissue and the responsiveness of DCs to IL-8 are a likely explanation of the clinical images, which suggest retention of DCs inside human malignant lesions. Impairment of DC migration toward lymphoid tissue could be involved in cancer immune evasion.
International Journal of Cancer 08/2005; 116(2):275-81. DOI:10.1002/ijc.21046 · 5.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gene delivery of IFN-alpha to the liver may represent an interesting strategy to maximize its antiviral efficacy and reduce side effects. We used a recombinant adeno-associated virus (AAV) encoding woodchuck IFN-alpha (AAV-IFN) to treat animals with chronic woodchuck hepatitis virus infection. The vector was given by intraportal or intramuscular route. Long-term transgene expression was detected after intraportal administration of an AAV encoding luciferase. In contrast, in the majority of the animals that received AAV-IFN through the portal vein, the expression of IFN-alpha was transient (30-40 days) and was associated with a significant but transient decrease in viral load. One animal, in which hepatic production of IFN-alpha persisted at high levels, died because of bone marrow toxicity. The disappearance of IFN-alpha expression correlated with the disappearance of AAV genomes from the liver. Intramuscular administration of AAV-IFN resulted in prolonged but fluctuating expression of the cytokine with no significant antiviral effect. In summary, this report shows that long-term expression of IFN-alpha in muscle is feasible but higher interferon levels might be needed to control viral replication. On the other hand, IFN-alpha gene delivery to the liver using an AAV vector induces a significant but transient antiviral effect in the woodchuck model.
[Show abstract][Hide abstract] ABSTRACT: In gene-therapy protocols, imaging of gene expression is needed to evaluate the transduction efficiency of the vector, its tissue distribution, and the duration of transgene expression and to assess the feasibility of repeated vector administration.
We have used positron emission tomography with a fluorine-18-labeled penciclovir analogue to monitor thymidine kinase gene expression after intratumoral injection of a first-generation recombinant adenovirus in patients with hepatocellular carcinoma. Patients were enrolled in a pilot clinical trial and treated with escalating doses of the vector. Two days after adenovirus inoculation, transgene expression was evaluated during the first hours after administration of the radiotracer both on the treated lesion and on a whole-body basis.
Transgene expression in the tumor was dependent on the injected dose of the adenovirus and was detectable in all patients who received > or = 10(12) viral particles. However, when the study was repeated 9 days after vector injection, no expression could be observed. It is interesting to note that no specific expression of the transgene could be detected in distant organs or in the surrounding cirrhotic tissue in any of the cases studied.
Our findings show the real possibility of imaging transgene expression in humans by using viral vectors. We show that hepatocarcinoma is a permissive tumor for adenoviral infection and that the nontumoral cirrhotic liver is spared from transduction when the vector is administered by intratumoral injection. These results show that positron emission tomography imaging may help in the design of gene-therapy strategies and in the clinical assessment of new-generation vectors.
[Show abstract][Hide abstract] ABSTRACT: The induction of protective or therapeutic cellular immunity against hepatitis C virus (HCV) is a difficult goal. In a previous work we showed that immunization with a recombinant adenovirus encoding HCV-NS3 (RAdNS3) could partially protect mice from challenge with a vaccinia virus encoding HCV antigens. We sought to investigate whether systemic administration of an immunostimulatory monoclonal antibody directed against the lymphocyte surface molecule CD137 could enhance the immunity elicited by RAdNS3. It was found that treatment with anti-CD137 mAb after the administration of a suboptimal dose of RAdNS3 enhanced cytotoxic and T helper cell responses against HCV NS3. Importantly, the ability of RAdNS3 to induce protective immunity against challenge with a recombinant vaccinia virus expressing HCV proteins was markedly augmented. Thus, combination of immunostimulatory anti-CD137 mAb with recombinant adenoviruses expressing HCV proteins might be useful in strategies of immunization against HCV.
[Show abstract][Hide abstract] ABSTRACT: To evaluate the feasibility and safety of intratumoral injection of autologous dendritic cells (DCs) transfected with an adenovirus encoding interleukin-12 genes (AFIL-12) for patients with metastatic gastrointestinal carcinomas. Secondarily, we have evaluated biologic effects and antitumoral activity.
Seventeen patients with metastatic pancreatic (n = 3), colorectal (n = 5), or primary liver (n = 9) malignancies entered the study. DCs were generated from CD14+ monocytes from leukapheresis, cultured and transfected with AFIL-12 before administration. Doses from 10 x 10(6) to 50 x 10(6) cells were escalated in three cohorts of patients. Patients received up to three doses at 21-day intervals.
Fifteen (88%) and 11 of 17 (65%) patients were assessable for toxicity and response, respectively. Intratumoral DC injections were mainly guided by ultrasound. Treatment was well tolerated. The most common side effects were lymphopenia, fever, and malaise. Interferon gamma and interleukin-6 serum concentrations were increased in 15 patients after each treatment, as well as peripheral blood natural killer activity in five patients. DC transfected with AFIL-12 stimulated a potent antibody response against adenoviral capsides. DC treatment induced a marked increase of infiltrating CD8+ T lymphocytes in three of 11 tumor biopsies analyzed. A partial response was observed in one patient with pancreatic carcinoma. Stable disease was observed in two patients and progression in eight patients, with two of the cases fast-progressing during treatment.
Intratumoral injection of DC transfected with an adenovirus encoding interleukin-12 to patients with metastatic gastrointestinal malignancies is feasible and well tolerated. Further studies are necessary to define and increase clinical efficacy.
[Show abstract][Hide abstract] ABSTRACT: The induction of IFN-gamma-secreting CD8+ T cells and neutralizing antibodies to HIV-1 are both key requirements for prevention of viral transmission and clearance of pathogenic HIV. Although DNA vaccination has been shown to induce both humoral and cellular immune responses against HIV antigens, the magnitude of the immune responses has always been disappointing. In this report, we analyze the ability of polyethylenimine (PEI)-DNA complex expressing an HIV-glycoprotein 120 (gp120) antigen (PEI-pgp120) to induce systemic CD8+ T cell and humoral responses to the gp120 antigen. The administration of PEI-plasmid complex resulted in rapid elevation of serum levels of IL-12 and IFN-gamma. Furthermore, a single administration of PEI-pgp120 complex elicits a number of gp120-specific CD8+ T cells 20 times higher than that elicited by three intramuscular injections of naked DNA. Interestingly, we found that systemic vaccination with PEI-pgp120 induced protective immune responses against both systemic and mucosal challenges with a recombinant vaccinia virus expressing a gp120 antigen. The data also demonstrated that the depletion of macrophages with liposome-encapsulated clodronate completely abolished gp120-specific cellular response. Overall, our results showed that a single administration of PEI-pgp120 complexes, eliciting strong immune responses, is an effective vaccination approach to generate protection against systemic and mucosal viral infections.
[Show abstract][Hide abstract] ABSTRACT: Quasispecies shifts are essential for the development of persistent hepatitis C virus (HCV) infection. Naturally occurring sequence variations in the 5' non-translated region (NTR) of the virus could lead to changes in protein expression levels, reflecting selective forces on the virus. The extreme 5' end of the virus' genome, containing signals essential for replication, is followed by an internal ribosomal entry site (IRES) essential for protein translation as well as replication. The 5' NTR is highly conserved and has a complex RNA secondary structure consisting of several stem-loops. This report analyses the quasispecies distribution of the 5' NTR of an HCV genotype 1b clinical isolate and found a number of sequences differing from the consensus sequence. The consensus sequence, as well as a major variant located in stem-loop IIIa of the IRES, was investigated using self-replicating HCV RNA molecules in human hepatoma cells. The stem-loop IIIa mutation, which is predicted to disrupt the stem structure, showed slightly lower translation efficiency but was severely impaired in the colony formation of selectable HCV replicons. Interestingly, during selection of colonies supporting autonomous replication, mutations emerged that restored the base pairing in the stem-loop. Recloning of these altered IRESs confirmed that these second site revertants were more efficient in colony formation. In conclusion, naturally occurring variants in the HCV 5' NTR can lead to changes in their replication ability. Furthermore, IRES quasispecies evolution was observed in vitro under the selective pressure of the replicon system.
Journal of General Virology 08/2004; 85(Pt 7):1859-66. DOI:10.1099/vir.0.79924-0 · 3.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To evaluate the feasibility and safety of intratumoral injection of an adenoviral vector encoding human interleukin-12 genes (Ad.IL-12) and secondarily, its biologic effect for the treatment of advanced digestive tumors.
Ad.IL-12 was administered in doses ranging from 2.5 x 10(10) to 3 x 10(12) viral particles, to seven cohorts of patients with advanced pancreatic, colorectal, or primary liver malignancies. Patients were thoroughly assessed for toxicity, and antitumor response was evaluated by imaging techniques, tumor biopsy, and hypersensitivity skin tests. Patients with stable disease and no serious adverse reactions were allowed to receive up to 3 monthly doses of Ad.IL-12.
Twenty-one patients (nine with primary liver, five with colorectal, and seven with pancreatic cancers) received a total of 44 injections. Ad.IL-12 was well tolerated, and dose-limiting toxicity was not reached. Frequent but transient adverse reactions, including fever, malaise, sweating, and lymphopenia, seemed to be related to vector injection rather than to transgene expression. No cumulative toxicity was observed. In four of 10 assessable patients, a significant increase in tumor infiltration by effector immune cells was apparent. A partial objective remission of the injected tumor mass was observed in a patient with hepatocellular carcinoma. Stable disease was observed in 29% of patients, mainly those with primary liver cancer.
Intratumoral injection of up to 3 x 10(12) viral particles of Ad.IL-12 to patients with advanced digestive malignancies is a feasible and well-tolerated procedure that exerts only mild antitumor effects.
[Show abstract][Hide abstract] ABSTRACT: Cellular immune response plays an important role in the clearance of hepatitis C virus (HCV). Thus, development of efficient ways to induce anti-viral cellular immune responses is an important step toward prevention and/or treatment of HCV infection. With this aim, we have constructed a replication-deficient recombinant adenovirus expressing HCV NS3 protein (RAdNS3). The efficacy of RAdNS3 was tested in vivo by measuring the protection against infection with a recombinant vaccinia virus expressing HCV-polyprotein (vHCV1-3011). Immunisation with 10(9)pfu of RAdNS3 induced anti-NS3 humoral, T helper and T cytotoxic responses. We identified eight epitopes recognised by IFN-gamma producing cells, five of them exhibiting lytic activity. Moreover, we show that RAdNS3 immunised mice were protected against challenge with vHCV1-3011 and that this protection was mediated by CD8(+) cells. In conclusion, our results suggest that adenoviral vectors encoding NS3 might be useful for the induction of prophylactic and/or therapeutic anti-HCV immunity.
[Show abstract][Hide abstract] ABSTRACT: Interferon-alpha (IFN-alpha) is a key element in the defense against viral infection because, in addition to a direct antiviral effect, it exhibits potent immunostimulatory activity. To investigate the function of this cytokine in the woodchuck model of chronic hepatitis B, the woodchuck IFN-alpha gene (IFNA) family was cloned and examined. The data indicate that this is a multigenic family from which 12 IFNA functional sequences and four pseudogene sequences were isolated. The overall identity of the amino acid sequence among the members of the woodchuck IFN-alpha family is 85%, and the identity with the IFN-alpha family from other species such as mice and humans is 50%. The analysis of hepatic expression of IFNA genes showed that wIFNA5a was the subtype transcribed preferentially in the woodchuck liver. The wIFNA genes transcribed in the liver were tested in an eukaryotic expression system and were found to enhance 2-5-oligoadenylate synthetase (2-5-OAS) mRNA levels and to posses a potent antiviral activity. Cloning of woodchuck IFNA genes will allow testing diverse forms of IFN-alpha delivery as well as different combination therapies in woodchuck hepatitis virus infection, thus providing useful information for the design of new strategies for the treatment of patients with chronic hepatitis B.
Journal of Medical Virology 04/2002; 68(3):424-32. DOI:10.1002/jmv.10221 · 2.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This review summarizes the status of gene therapy in medicine and the role of molecular imaging in its development. In gene therapy, genetic material is introduced into cells in order to generate a specific biological effect. Natural (viruses) or artificial molecular constructs, named gene therapy vectors, are used to achieve efficient cell transduction. This new form of therapy can be used for treating a broad variety of conditions including hereditary diseases, infections, degenerative disorders and cancer. Monitoring transgene expression using noninvasive imaging techniques is a necessary complement for the development of clinical gene therapy. Recent developments in magnetic resonance imaging afford the possibility of detecting gene transfer in vivo, but the most promising results have been obtained with positron emission tomography (PET). PET allows imaging gene therapy products by administration of a labeled substrate when the transgene codes for an enzyme or by administration of a labeled ligand when the transgene codes for a receptor. In the latter strategy, a membrane molecule (somatostatin or dopamine receptors) is used to detect the selective trapping of its radiolabeled ligand in the transduced cells. One of the approaches for the genetic treatment of cancer consists in transferring the "suicide genes" into tumor cells, the most common being the thymidine kinase (tk) of herpes viruses. Different nucleoside analogs can be labeled for its use as PET reporter probes in order to visualize tk expression. The results of pre-clinical studies are extremely encouraging. Reliable methods for the in vivo tracing of transgene expression in humans have to be developed in order for the field of gene therapy to mature. PET has emerged as a powerful tool to assist in achieving this goal.
[Show abstract][Hide abstract] ABSTRACT: This review summarizes the status of gene therapy in medicine and the role of molecular imaging in its development. In gene therapy, genetic material is introduced into cells in order to generate a specific biological effect. Natural (viruses) or artificial molecular constructs, named gene therapy vectors, are used to achieve efficient cell transduction. This new form of therapy can be used for treating a broad variety of conditions including hereditary diseases, infections, degenerative disorders and cancer. Monitoring transgene expression using noninvasive imaging techniques is a necessary complement for the development of clinical gene therapy. Recent developments in magnetic resonance imaging afford the possibility of detecting gene transfer in vivo, but the most promising results have been obtained with positron emission tomography (PET). PET allows imaging gene therapy products by administration of a labeled substrate when the transgene codes for an enzyme or by administration of a labeled ligand when the transgene codes for a receptor. In the latter strategy, a membrane molecule (somatostatin or dopamine receptors) is used to detect the selective trapping of its radiolabeled ligand in the transduced cells. One of the approaches for the genetic treatment of cancer consists in transferring the “suicide genes” into tumor cells, the most common being the thymidine kinase (tk) of herpes viruses. Different nucleoside analogs can be labeled for its use as PET reporter probes in order to visualize tk expression. The results of pre-clinical studies are extremely encouraging. Reliable methods for the in vivo tracing of transgene expression in humans have to be developed in order for the field of gene therapy to mature. PET has emerged as a powerful tool to assist in achieving this goal. (Mol Imag Biol 2002;4:27–33)