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3.
Orphanet J Rare Dis ; 15(1): 341, 2020 12 03.
Article in English | MEDLINE | ID: mdl-33272301

ABSTRACT

The ongoing coronavirus disease 2019 (COVID-19) pandemic has caused disruption in all aspects of daily life, including the management and treatment of rare inherited metabolic disorders (IMDs). To perform a preliminary assessment of the incidence of COVID-19 in IMD patients and the impact of the coronavirus emergency on the rare metabolic community between March and April 2020, the European Reference Network for Hereditary Metabolic Diseases (MetabERN) has performed two surveys: one directed to patients' organizations (PO) and one directed to healthcare providers (HCPs). The COVID-19 incidence in the population of rare metabolic patients was lower than that of the general European population (72.9 × 100,000 vs. 117 × 100,000). However, patients experienced extensive disruption of care, with the majority of appointments and treatments cancelled, reduced, or postponed. Almost all HCPs (90%) were able to substitute face-to-face visits with telemedicine, about half of patients facing treatment changes switched from hospital to home therapy, and a quarter reported difficulties in getting their medicines. During the first weeks of emergency, when patients and families lacked relevant information, most HCPs contacted their patients to provide them with support and information. Since IMD patients require constant follow-up and treatment adjustments to control their disease and avoid degradation of their condition, the results of our surveys are relevant for national health systems in order to ensure appropriate care for IMD patients. They highlight strong links in an interconnected community of HCPs and PO, who are able to work quickly and effectively together to support and protect fragile persons during crisis. However, additional studies are needed to better appreciate the actual impact of COVID-19 on IMD patients' health and the mid- and long-term effects of the pandemic on their wellbeing.


Subject(s)
COVID-19/complications , Metabolic Diseases/complications , Rare Diseases/complications , SARS-CoV-2 , COVID-19/epidemiology , Data Collection , Europe/epidemiology , Genetic Predisposition to Disease , Health Personnel , Health Services Accessibility , Humans , Metabolic Diseases/classification , Telemedicine
4.
J Virol ; 75(22): 11166-77, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11602756

ABSTRACT

Whereas human immunodeficiency virus (HIV) infects various cell types by fusion at the plasma membrane, we observed a different entry route in human primary macrophages, in which macropinocytosis is active. Shortly after exposure of macrophages to HIV-1 and irrespective of viral envelope-receptor interactions, particles were visible in intracellular vesicles, which were identified as macropinosomes. Most virions appeared subsequently degraded. However, fusion leading to capsid release in the cytosol and productive infection could take place inside vesicles when particles were properly enveloped. These observations provide new insights into HIV-1 interactions with a cell target relevant to pathogenesis. They may have implications for the design of soluble inhibitors aimed at interfering with the fusion or entry processes.


Subject(s)
HIV-1/physiology , Macrophages/virology , Pinocytosis , Cytosol/chemistry , HIV Core Protein p24/analysis , HeLa Cells , Humans , Hydrogen-Ion Concentration , Macrophages/ultrastructure , Microscopy, Electron
5.
Mol Ther ; 4(2): 149-56, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11482987

ABSTRACT

We investigated the production efficiency and the gene transfer capacity in the central nervous system of HIV-1-based vectors pseudotyped with either the G protein of the Mokola lyssaviruses (MK-G), a neurotropic virus causing rabies disease, or the vesiculo-stomatitis G protein (VSV-G). Both envelopes induced syncitia in cell cultures. They were incorporated into vector particles and mature virions were observed by electron microscopy. Vector production was two- to sixfold more efficient with VSV-G than with MK-G. For equivalent amounts of physical particles, vector titration was 5- to 25-fold higher with VSV-G than with MK-G pseudotypes on cultured cells, and in vivo gene expression in mouse brain was more intense. Thus, VSV-G pseudotypes were produced more efficiently and were more infectious than MK-G pseudotypes. Tropism for brain cells was analyzed by intrastriatal injections in rats. Both pseudotypes preferentially transduced neurons (70-90% of transduced cells). Retrograde axonal transport was investigated by instilling vector suspensions in the rat nasal cavity. Both pseudotypes were efficiently transported to olfactive neuron bodies. Thus, although coating HIV-1 particles with rabdhovirus envelope glycoproteins enables them to enter neuronal cells efficiently, pseudotyping is not sufficient to confer the powerful neurotropism of lyssaviruses to lentivirus vectors.


Subject(s)
Brain/physiology , Genetic Vectors , HIV-1/genetics , Lyssavirus/genetics , Membrane Glycoproteins , Viral Envelope Proteins/genetics , Animals , Brain/cytology , Brain/virology , Cell Line , Corpus Striatum , Gene Transfer Techniques , Glucuronidase/genetics , Glucuronidase/metabolism , HIV-1/physiology , Humans , Injections , Lyssavirus/physiology , Mice , Microscopy, Fluorescence , Neurons/physiology , Rats , Transfection , Viral Envelope Proteins/metabolism , beta-Galactosidase/genetics , beta-Galactosidase/metabolism
6.
Virology ; 286(1): 225-36, 2001 Jul 20.
Article in English | MEDLINE | ID: mdl-11448175

ABSTRACT

Dendritic cells (DCs) are thought to play a crucial role in the pathogenesis of HIV-1 infection. DCs are believed to transport virus particles to lymph nodes before transfer to CD4(+) lymphocytes. We have investigated the role of Nef in these processes. HIV-1 replication was examined in human immature DC-lymphocyte cocultures and in DCs or lymphocytes separately. Using various R5-tropic and X4-tropic HIV-1 strains and their nef-deleted (Deltanef) counterparts, we show that Nef is required for optimal viral replication in immature DC-T cells clusters and in T lymphocytes. Nef exerts only a marginal role on viral replication in immature DCs alone as well as on virion capture by DCs, long-term intracellular accumulation and transmission of X4 strains to lymphocytes. We also show that wild-type and Deltanef virions are similarly processed for MHC-I restricted exogenous presentation by DCs. Taken together, these results help explain how HIV-1 Nef may affect viral spread and immune responses in the infected host.


Subject(s)
Dendritic Cells/virology , Gene Products, nef/physiology , HIV Infections/virology , HIV-1/physiology , Lymphocytes/virology , Cell Communication , Cells, Cultured , Coculture Techniques , Dendritic Cells/pathology , HIV Infections/pathology , Humans , Lymphocytes/pathology , Virus Replication , nef Gene Products, Human Immunodeficiency Virus
7.
J Virol ; 75(12): 5584-92, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11356966

ABSTRACT

PiT-1 and PiT-2 are related multiple transmembrane proteins which function as sodium-dependent phosphate transporters and as the cell receptors of several oncoretroviruses. Two copies of a homology domain that is found in distantly related species assign these proteins to a large family of phosphate transporters. A current membrane topology model of PiT-1 and PiT-2 predicts 10 transmembrane domains. However, the validity of this model has not been addressed experimentally. We addressed this issue by a comprehensive study of human PiT-2. Evidence was obtained for glycosylation of asparagine 81. Epitope tagging showed that the N- and C-terminal extremities are extracellular. The orientation of C-terminal-truncation mutants expressed in cell-free translation assays and incorporated into microsomal membranes was examined by immunoprecipitation. Data were interpreted with respect to previous knowledge about retrovirus binding sites, to the existence of repeated homology domains, and to predictions made in family members. A model in which PiT-2 has 12 transmembrane domains and extracellular N- and C-terminal extremities is proposed. This model, which differs significantly from previous predictions about PiT-2 topology, may be useful for further investigations of PiT-2 interactions with other proteins and for the understanding of PiT-2 transporter and virus receptor functions.


Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , Receptors, Virus/metabolism , Retroviridae/metabolism , Symporters , Amino Acid Sequence , Animals , Asparagine/metabolism , Computational Biology , Epitopes , Glycosylation , Humans , Mice , Models, Biological , Molecular Sequence Data , Mutation , Precipitin Tests , Protein Biosynthesis , Rats , Sodium-Phosphate Cotransporter Proteins , Sodium-Phosphate Cotransporter Proteins, Type III
8.
Blood ; 97(8): 2213-20, 2001 Apr 15.
Article in English | MEDLINE | ID: mdl-11290581

ABSTRACT

Mechanisms governing the induction of effective erythropoiesis in response to erythropoietin (Epo) oversecretion have been investigated in beta thalassemic C57Bl/6(Hbbth) mice. Naked DNA encoding an expression vector for mouse Epo was introduced into skeletal muscles by electrotransfer. A transient increase of serum Epo concentrations with a proportional augmentation of hematocrit values was observed. Various parameters relevant to beta thalassemia were surveyed in blood samples taken before treatment, at the peak of Epo secretion, and when the phenotype reverted to anemia. We measured globin messenger RNA (mRNA) levels in reticulocytes by real-time quantitative polymerase chain reaction, globin chain synthesis levels, and several indicators of erythrocyte membrane quality, including bound alpha chains, bound immunoglobulins, main protein components, and iron compartmentalization. Data indicated that high serum Epo levels primarily affect betaminor-globin mRNA accumulation in reticulocytes. Other changes subsequent to intense Epo stimulation, like increased betaminor/alpha-globin chain synthesis ratio, reduced levels of alpha chains and immunoglobulins bound to membranes, improved spectrin/band 3 ratio, increased red blood cell survival, and improved erythropoiesis appeared as consequences of increased betaminor-globin mRNA levels. This conclusion is consistent with models postulating that intense Epo stimulation induces the expansion and differentiation of erythroid progenitors committed to fetal erythropoiesis. Although phenotypic correction was partial in mice, and comparable achievements will probably be more difficult to obtain in humans, naked DNA electrotransfer may provide a safe and low-cost method for reassessing the potentials of Epo as an inducer of fetal erythropoiesis reactivation in patients with beta thalassemia.


Subject(s)
DNA, Complementary/genetics , Erythropoiesis/genetics , Erythropoietin/genetics , Globins/genetics , Muscle, Skeletal/metabolism , RNA, Messenger/biosynthesis , Reticulocytes/metabolism , beta-Thalassemia/genetics , Animals , Anion Exchange Protein 1, Erythrocyte/analysis , Cell Compartmentation , Cell Differentiation , DNA, Complementary/administration & dosage , Disease Models, Animal , Electroporation , Erythrocytes/metabolism , Erythroid Precursor Cells/metabolism , Erythroid Precursor Cells/pathology , Erythropoietin/blood , Genetic Complementation Test , Injections, Intramuscular , Iron/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Polymerase Chain Reaction , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/physiology , Spectrin/analysis , Transfection
9.
Nat Med ; 7(3): 344-9, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11231634

ABSTRACT

Dendritic cells and macrophages can process extracellular antigens for presentation by MHC-I molecules. This exogenous pathway may have a crucial role in the activation of CD8+ cytotoxic T lymphocytes during human viral infections. We show here that HIV-1 epitopes derived from incoming virions are presented through the exogenous MHC-I pathway in primary human dendritic cells, and to a lower extent in macrophages, leading to cytotoxic T-lymphocyte activation in the absence of viral protein synthesis. Exogenous antigen presentation required adequate virus-receptor interactions and fusion of viral and cellular membranes. These results provide new insights into how anti-HIV cytotoxic T lymphocytes can be activated and have implications for anti-HIV vaccine design.


Subject(s)
HIV Antigens/immunology , HIV-1/immunology , Histocompatibility Antigens Class I/immunology , Virion/immunology , Virus Replication , Cell Line , Cross Reactions , Epitopes/immunology , HIV-1/physiology , Humans
10.
J Mol Cell Cardiol ; 33(2): 295-305, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11162134

ABSTRACT

Vascular endothelial growth factor (VEGF) has proven to be one of the most effective growth factors for therapeutic angiogenesis. The biological efficacy of the adeno-associated virus (AAV) vector has recently been demonstrated in muscle tissues, including the heart. Apart from these promising insights into VEGF and the AAV vector, studies on VEGF gene transfer using the AAV vector have been limited. Here, we evaluate AAV-mediated VEGF gene transfer, both in vitro and in vivo, using the AAV-mVEGF vector that contains cDNA for murine VEGF(120) within an HCMV-driven expression cassette. Transient transfection of AAV-mVEGF plasmid significantly increased mVEGF expression in 293T cells. The secreted VEGF in the conditioned medium had strong biological activity, as confirmed by the Miles' vascular permeability assay. Transduction of 293T and HeLa cells with AAV-mVEGF stock of high titer, that is essentially adenovirus-free, showed significantly increased mVEGF expression above that of AAV-eGFP-transduced cells. When human umbilical vein endothelial cells were transduced, a higher level of mVEGF expression, together with higher cell counts, was observed compared to AAV-eGFP-transduced cells. In vivo transduction of mouse tibialis anterior muscle resulted in an increased level of mVEGF expression, and higher capillary-to-myofibre ratio, 8 weeks post-transduction. In a rat hindlimb ischemia model, regional blood flow, as well as the capillary-to-myofibre ratio, was significantly increased at 4 weeks post-transduction. These findings demonstrate the efficient delivery of the VEGF gene using an AAV vector, which has implications for angiogenic gene therapy in ischemic diseases.


Subject(s)
Dependovirus/genetics , Endothelial Growth Factors/biosynthesis , Endothelial Growth Factors/genetics , Gene Transfer Techniques , Genetic Vectors , Lymphokines/biosynthesis , Lymphokines/genetics , Animals , Capillary Permeability , Cell Line , Cells, Cultured , Culture Media, Conditioned/pharmacology , DNA, Complementary/metabolism , Endothelium, Vascular/cytology , Extremities/blood supply , Female , Guinea Pigs , HeLa Cells , Humans , Ischemia/metabolism , Male , Mice , Mice, Inbred BALB C , Models, Genetic , Neovascularization, Physiologic , Rats , Rats, Sprague-Dawley , Regional Blood Flow , Time Factors , Transduction, Genetic , Transfection , Umbilical Veins/cytology , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth Factors
11.
J Am Soc Nephrol ; 11 Suppl 16: S159-62, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11065350

ABSTRACT

Erythropoietin (Epo) is a glycoprotein hormone produced by genetic engineering. Many pathologic conditions could benefit from its administration, such as chronic renal failure or hemoglobinopathies. Epo secretion from genetically modified tissued could be proposed to patients only if the protocol is low cost and low risk. For that purpose, retroviral vectors and adeno-associated vectors expressing the Epo cDNA were developed. Gene transfer was performed into skeletal muscles. To avoid polycythemia, a tetracycline-regulated system was used to control the levels of protein secretion in vivo. beta-thalassemias are among diseases that could benefit from an Epo gene transfer. beta-thalassemias are attributable to deficient synthesis of beta-globin and accumulation of unpaired alpha-chains. Stimulation of fetal globin synthesis is one strategy to correct the globin chain imbalance. There is evidence that Epo could play this role. In a mouse model of beta-thalassemia, an adeno-associated vector expressing the Epo cDNA was injected intramuscularly. Epo was secreted continuously during at least 1 yr. Erythropoiesis was improved in those mice by increasing the synthesis of fetal hemoglobin.


Subject(s)
Cell Transplantation , Erythropoietin/administration & dosage , Genetic Engineering , Animals , Erythropoietin/genetics , Erythropoietin/therapeutic use , Gene Transfer Techniques , Genetic Therapy , Humans , beta-Thalassemia/therapy
12.
J Virol ; 74(19): 9256-66, 2000 Oct.
Article in English | MEDLINE | ID: mdl-10982373

ABSTRACT

The human immunodeficiency virus type 1 Nef protein alters the post-Golgi stages of major histocompatibility complex class I (MHC-I) biogenesis. Presumed mechanisms involve the disclosure of a cryptic tyrosine-based sorting signal (YSQA) located in the cytoplasmic tail of HLA-A and -B heavy chains. We changed this signal for a prototypic sorting motif (YSQI or YSQL). Modified HLA-A2 molecules, termed A2-endo, displayed constitutively low surface levels and accumulated in a region close to or within the Golgi apparatus, a behavior reminiscent of wild-type HLA-A2 in Nef-expressing cells. However, several lines of evidence indicate that the action of prototypic signals on MHC-I trafficking differs from that of Nef. Internalization of surface A2-endo was more rapid and was associated with efficient recycling to the surface. A transdominant-negative mutant of dynamin-1 inhibited A2-endo constitutive internalization and Nef-induced CD4 down-regulation, whereas it did not affect the activity of Nef on MHC-I. Moreover, trafficking of A2-endo was still affected by the viral protein, indicating additive effects of prototypic signals and Nef. Therefore, distinct trafficking pathways regulate clathrin-dependent and Nef-induced MHC-I modulation.


Subject(s)
Clathrin/physiology , Gene Products, nef/physiology , HIV-1/physiology , Histocompatibility Antigens Class I/physiology , Down-Regulation , Golgi Apparatus/physiology , HeLa Cells , Humans , Signal Transduction , Virus Replication , nef Gene Products, Human Immunodeficiency Virus
13.
Mol Ther ; 1(1): 63-70, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10933913

ABSTRACT

Most lysosomal storage diseases, including mucopolysaccharidosis, affect the central nervous system (CNS). They often induce severe and progressive mental retardation. Replacement therapy by purified enzyme infusions is a promising approach for the treatment of peripheral organs but without effect on CNS pathology because the enzyme cannot cross the blood-brain barrier. Intracranial injection of recombinant adeno-associated virus (AAV) vectors offers an alternative for sustained local enzyme delivery from genetically engineered cells. We stereotactically injected an AAV vector containing the human beta-glucuronidase cDNA into the striatum of adult mice severely affected by mucopolysaccharidosis type VII at the time of treatment. Six weeks later, beta-glucuronidase activity in the injected hemisphere was comparable to that of heterozygous mice, which have a normal phenotype. Areas staining positive for enzyme activity enlarged with time, representing more than 10% of the hemisphere volume by 16 weeks. A complete reversion of lysosomal storage lesions was evident in these areas, as well as in most neurons located in surrounding negative areas and in the noninjected hemisphere. Thus, a single intracerebral injection of AAV vectors could achieve a broad and sustained lysosomal enzyme delivery, allowing for stable reversion of storage lesions in a significant fraction of the adult brain.


Subject(s)
Brain/enzymology , Dependovirus/genetics , Genetic Therapy/methods , Glucuronidase/genetics , Mucopolysaccharidosis VII/enzymology , Mucopolysaccharidosis VII/therapy , Animals , Brain/pathology , DNA, Complementary/administration & dosage , DNA, Complementary/genetics , Gene Expression , Genetic Vectors , Glucuronidase/metabolism , Histocytochemistry , Humans , In Situ Hybridization , Injections , Mice , Mice, Mutant Strains , Mucopolysaccharidosis VII/pathology , RNA, Messenger/genetics , RNA, Messenger/metabolism
14.
Hum Gene Ther ; 11(8): 1139-50, 2000 May 20.
Article in English | MEDLINE | ID: mdl-10834616

ABSTRACT

Gene transfer vectors derived from human immunodeficiency virus (HIV-1) efficiently transduce nondividing cells and remain stably integrated in their genome. Long-term expression of reporter genes has been documented after intracerebral injection of these vectors. Using a HIV-based vector, we looked for a reversal of brain damage in the beta-glucuronidase-deficient mucopolysaccharidosis type VII mouse, an animal model of human lysosomal storage diseases. The vector suspension was injected stereotactically in the brain of 10-week-old animals, an age at which storage lesions are patent in glia, perivascular cells, and neurons. Either a single intrastriatal injection or multiple injections in both cerebral hemispheres and in the cerebellum were performed. Local tolerance, enzyme delivery, and correction of storage lesions were investigated by comprehensive analysis of serial sections of the entire brain of mice killed 6 or 16 weeks postinjection. Histochemical staining detected enzyme activity in widely distributed areas, the size of which increased with time. Clearance of lysosomal storage extended far beyond enzyme-positive areas. In mice receiving multiple injections of the vector, complete correction or significant reduction of the pathology was observed in every section, suggesting disease regression in the entire brain. These results may have implications for the treatment of neurological symptoms in lysosomal storage diseases.


Subject(s)
Brain/pathology , Gene Transfer Techniques , Glucuronidase/genetics , HIV/genetics , Lentivirus/genetics , Mucopolysaccharidosis VII/pathology , Mucopolysaccharidosis VII/therapy , Animals , Brain/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , Glucuronidase/metabolism , In Situ Hybridization , Mice , Plasmids/genetics , Polymerase Chain Reaction , Time Factors , Tissue Distribution , beta-Galactosidase/genetics , beta-Galactosidase/metabolism , beta-N-Acetylhexosaminidases/genetics , beta-N-Acetylhexosaminidases/metabolism
15.
Blood ; 95(9): 2793-8, 2000 May 01.
Article in English | MEDLINE | ID: mdl-10779423

ABSTRACT

beta-Thalassemias are highly prevalent genetic disorders that can cause severe hemolytic anemia. The main pathophysiologic feature of beta-thalassemia is the accumulation of unpaired alpha-globin chains in erythrocyte precursors and red blood cells (RBCs). This accumulation alters cell membrane function and results in early cell destruction and ineffective erythropoiesis. Correction of globin chain imbalance through the induction of fetal hemoglobin (HbF) synthesis is a tentative therapeutic approach for this class of diseases. In short-term in vitro or in vivo assays, recombinant human erythropoietin increases the frequency of erythroid precursors programmed to HbF in humans and to beta-minor globin in mice. In contrast, long-term treatment of beta-thalassemic patients did not induce HbF significantly. We took advantage of highly efficient adeno-associated virus-mediated (AAV-mediated) gene transfer into mouse muscle to induce a robust and sustained secretion of mouse erythropoietin in beta-thalassemic mice, which represent a suitable model for human beta-thalassemia intermedia. A 1-year follow-up of 12 treated animals showed a stable correction of anemia associated with improved RBC morphology, increased beta-minor globin synthesis, and decreased amounts of alpha-globin chains bound to erythrocyte membranes. More effective erythropoiesis probably accounted for a reduction of erythroid cell proliferation, as shown by decreased proportions of circulating reticulocytes and by reduced iron 59 ((59)Fe) incorporation into erythroid tissues. This study indicates that the continuous delivery of high amounts of autologous erythropoietin induced a sustained stimulation of beta-minor globin synthesis and a stable improvement of erythropoiesis in the beta-thalassemic mouse model. (Blood. 2000;95:2793-2798)


Subject(s)
Erythropoiesis , Erythropoietin/genetics , Genetic Therapy , beta-Thalassemia/therapy , Animals , Cell Division , Dependovirus , Erythrocyte Count , Erythropoietin/physiology , Gene Transfer Techniques , Genetic Vectors , Globins/biosynthesis , Hematocrit , Humans , Iron/metabolism , Mice , Mice, Mutant Strains , Muscle, Skeletal , Reticulocyte Count , beta-Thalassemia/blood
16.
Arterioscler Thromb Vasc Biol ; 20(2): 435-42, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10669641

ABSTRACT

The apolipoprotein E (apoE)-deficient mouse is a relevant animal model of human atherosclerosis. Although the prevention of atherosclerosis development has been documented after somatic gene transfer into animal models, regression of lesions remains to be demonstrated. Thus, we used this genetically defined mouse model nn the nude background to show atherosclerosis regression. ApoE-deficient nude mice were infected with 5 x 10(8) or 10(9) plaque-forming units of a first-generation adenovirus encoding human apoE cDNA. The secretion of human apoE resulted in a rapid decrease of total cholesterol, which normalized the hypercholesterolemic phenotype within 14 days (from 600+/-100 to <100 microg/mL). Transgene expression was observed during a period of >4 months, with a normalization of cholesterol and triglyceride levels during 5 months. At that time, we successfully reinjected the recombinant adenovirus and observed the appearance of the human protein as well as the correction of lipoprotein phenotype. In mice killed 6 months-after the first infection, we observed a dose-dependent regression of fatty streak lesions in the aorta. We showed sustained expression of a transgene with a first-generation adenoviral vector and a correction of dyslipoproteinemia phenotype leading to lesion regression. These data demonstrate that somatic gene transfer can induce plaque regression.


Subject(s)
Apolipoproteins E/deficiency , Apolipoproteins E/genetics , Arteriosclerosis/metabolism , Arteriosclerosis/pathology , Gene Transfer Techniques , Animals , Apolipoproteins E/metabolism , Arteriosclerosis/genetics , Humans , Lipoproteins/blood , Mice , Mice, Inbred C57BL , Mice, Nude , Mice, Transgenic/genetics , Tissue Distribution
17.
Biochem Biophys Res Commun ; 270(2): 637-42, 2000 Apr 13.
Article in English | MEDLINE | ID: mdl-10753676

ABSTRACT

Atrial natriuretic peptide (ANP) is a cardiac hormone that elicits a profound diuresis, natriuresis, and hypotension. As a preliminary study toward ANP gene therapy of cardiovascular disorders, we have cloned a cDNA for mouse preproANP and carried out expression studies in muscle cells. The expression cassette, which was flanked by ITRs from AAV-2, consisted of HCMV IE enhancer/promoter, preproANP gene, and polyadenylation signal from bovine growth hormone. We transfected this expression vector into primary skeletal myoblasts and examined the following points: (1) secretion of immunoreactive ANP, (2) biological activity, and (3) nature of secreted ANP(s). The conditioned media from cells transfected with ANP vector had significantly higher levels of irANP in comparison to mock control. The secreted irANP had biological activity as confirmed by the elevated level of intracellular cGMP in human umbilical vein endothelial cells. Reverse-phase HPLC analysis showed that the processed form of ANP was the predominant form. These results demonstrate that preproANP gene could be ectopically expressed and correctly processed in skeletal myoblasts, which has implications for development of muscle-based ANP gene therapy.


Subject(s)
Atrial Natriuretic Factor/metabolism , Muscle, Skeletal/metabolism , Protein Precursors/metabolism , Amino Acid Sequence , Animals , Atrial Natriuretic Factor/chemistry , Atrial Natriuretic Factor/genetics , Base Sequence , Cattle , Cloning, Molecular , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Molecular Sequence Data , Muscle, Skeletal/cytology , Protein Precursors/chemistry , Protein Precursors/genetics , Protein Processing, Post-Translational , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid
18.
J Virol ; 73(5): 3608-15, 1999 May.
Article in English | MEDLINE | ID: mdl-10196252

ABSTRACT

The alpha-chemokine SDF-1 binds CXCR4, a coreceptor for human immunodeficiency virus type 1 (HIV-1), and inhibits viral entry mediated by this receptor. Since chemokines are potent chemoattractants and activators of leukocytes, we examined whether the stimulation of HIV target cells by SDF-1 affects the replication of virus with different tropisms. We observed that SDF-1 inhibited the entry of X4 strains and increased the infectivity of particles bearing either a CCR5-tropic HIV-1 envelope or a vesicular stomatitis virus G envelope. In contrast to the inhibitory effect of SDF-1 on X4 strains, which is at the level of entry, the stimulatory effect does not involve envelope-receptor interactions or proviral DNA synthesis. Rather, we observed an increased ability of Tat to transactivate the HIV-1 long terminal repeat in the presence of the chemokine. Therefore, the effects of SDF-1 on the HIV-1 life cycle can be multiple and opposite, including both an inhibition of viral entry and a stimulation of proviral gene expression.


Subject(s)
Anti-HIV Agents/pharmacology , Chemokines, CXC/pharmacology , HIV-1/drug effects , Virus Replication , Chemokine CXCL12 , Gene Products, tat/metabolism , HIV-1/metabolism , HIV-1/physiology , HeLa Cells , Humans , Lymphocytes/virology , Monocytes/virology , Transcription, Genetic , tat Gene Products, Human Immunodeficiency Virus
19.
J Virol ; 73(5): 3789-99, 1999 May.
Article in English | MEDLINE | ID: mdl-10196273

ABSTRACT

PIT-2 is a type III sodium phosphate cotransporter and the receptor for amphotropic murine leukemia viruses. We have investigated the expression and the functions of a tagged version of PIT-2 in CHO cells. PIT-2 remained equally abundant at the cell surface within 6 h following variation of the phosphate supply. In contrast, the efficiency of phosphate uptake and retrovirus entry was inversely related to the extracellular phosphate concentration, indicating that PIT-2 activities are modulated by posttranslational modifications of cell surface molecules induced by phosphate. Conformational changes of PIT-2 contribute to both activities, as shown by the inhibitory effect of sulfhydryl reagents known as inhibitors of type II cotransporters. A physical association of PIT-2 with actin was demonstrated. Modifications of the actin network were induced by variations of the concentrations of extracellular phosphate, cytochalasin D, or lysophosphatidic acid. They revealed that the formation of actin stress fibers determines the cell surface distribution of PIT-2, the internalization of the receptor in response to virus binding, and the capacity to process retrovirus entry. Thus, the presence of PIT-2 at the cell surface is not sufficient to ensure phosphate transport and susceptibility to amphotropic retrovirus infection. Further activation of cell surface PIT-2 molecules is required for these functions.


Subject(s)
Carrier Proteins/metabolism , Leukemia Virus, Murine/metabolism , Phosphates/metabolism , Protein Processing, Post-Translational , Receptors, Virus/metabolism , Symporters , Actins/metabolism , Animals , CHO Cells , Cricetinae , Humans , Mice , Sodium-Phosphate Cotransporter Proteins , Sodium-Phosphate Cotransporter Proteins, Type II , Sodium-Phosphate Cotransporter Proteins, Type III , Virion/metabolism
20.
Hum Gene Ther ; 10(3): 375-83, 1999 Feb 10.
Article in English | MEDLINE | ID: mdl-10048390

ABSTRACT

Cell encapsulation offers a safe and manufacturable method for the systemic delivery of therapeutic proteins from genetically engineered cells. However, control of dose delivery remains a major issue with regard to clinical application. We generated populations of immortalized murine NIH 3T3 fibroblasts that secrete mouse erythropoietin (Epo) in response to stimulation by doxycycline or mifepristone. Engineered cells were introduced into AN69 hollow fibers, which were implanted in the peritoneal cavity or recipient mice. Animals receiving doxycycline or mifepristone showed stable polyhemia and increased serum Epo concentrations over a 6-month observation period, whereas animals not receiving the inducer drug had normal hematocrits. Epo secretion could be switched on and off, depending on the presence of doxycycline in the drinking water. In contrast, polyhemia was hardly reversible after subcutaneous injections of mifepristone. These data show that a permanent and regulated systemic delivery of a therapeutic protein can be obtained by the in vivo implantation of engineered allogeneic cells immunoprotected in membrane polymers.


Subject(s)
Doxycycline/pharmacology , Erythropoietin/metabolism , Mifepristone/pharmacology , 3T3 Cells , Animals , Blotting, Southern , Capsules , Dose-Response Relationship, Drug , Doxycycline/administration & dosage , Erythropoietin/blood , Gene Expression Regulation , Gene Transfer Techniques , Genetic Therapy/methods , Hematocrit , Mice , Mifepristone/administration & dosage , Retroviridae , Time Factors
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