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1.
Gene Ther ; 8(16): 1207-13, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11509953

ABSTRACT

The development of therapies aimed to promote remyelination is a major issue in chronic inflammatory demyelinating disorders of the central nervous system (CNS) such as multiple sclerosis (MS), where the permanent neurological impairment is due to the axonal loss resulting from recurrent episodes of immune-mediated demyelination. Here, we show that the intrathecal injection of a herpes simplex virus (HSV) type-1 replication-defective multigene vector, engineered with the human fibroblast growth factor (FGF)-II gene (TH:bFGF vector), was able to significantly revert in C57BL/6 mice the clinicopathological signs of chronic experimental autoimmune encephalomyelitis (EAE), the animal model of MS. The treatment with the TH:bFGF vector was initiated within 1 week after the clinical onset of EAE and was effective throughout the whole follow-up period (ie 60 days). The disease-ameliorating effect in FGF-II-treated mice was associated with: (1) CNS production of FGF-II from vector-infected cells which were exclusively located around the CSF space (ependymal, choroidal and leptomeningeal cells); (2) significant decrease (P < 0.01) of the number of myelinotoxic cells (T cells and macrophages) both in the CNS parenchyma and in the leptomeningeal space; and (3) significant increase (P < 0.01) of the number of oligodendrocyte precursors and of myelin-forming oligodendrocytes in areas of demyelination and axonal loss. Our results indicate that CNS gene therapy using HSV-1-derived vector coding for neurotrophic factors (ie FGF-II) is a safe and non-toxic approach that might represent a potential useful 'alternative' tool for the future treatment of immune-mediated demyelinating diseases.


Subject(s)
Encephalomyelitis, Autoimmune, Experimental/therapy , Fibroblast Growth Factor 2/genetics , Genetic Therapy/methods , Animals , Axons/pathology , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/pathology , Female , Genetic Vectors/administration & dosage , Injections, Spinal , Lymphocyte Count , Macrophages/immunology , Mice , Mice, Inbred C57BL , Multiple Sclerosis/therapy , Oligodendroglia/pathology , Simplexvirus/genetics , Spinal Cord/immunology , Spinal Cord/pathology , T-Lymphocytes/immunology
2.
Hum Gene Ther ; 12(8): 905-20, 2001 May 20.
Article in English | MEDLINE | ID: mdl-11387056

ABSTRACT

Systemic administration of antiinflammatory molecules to patients affected by immune-mediated inflammatory demyelinating diseases of the central nervous system (CNS) has limited therapeutic efficacy due to the presence of the blood-brain barrier (BBB). We found that three of five rhesus monkeys injected intrathecally with a replication-defective herpes simplex virus (HSV) type 1-derived vector engineered with the human interleukin 4 (IL-4) gene were protected from an hyperacute and lethal form of experimental autoimmune encephalomyelitis induced by whole myelin. The intrathecally injected vector consistently diffused within the CNS via the cerebrospinal fluid and infected ependymal cells, which in turn sustained in situ production of IL-4 without overt immunological or toxic side effects. In EAE-protected monkeys, IL-4-gene therapy significantly decreased the number of brain as well as spinal cord inflammatory perivenular infiltrates and the extent of demyelination, necrosis, and axonal loss. The protective effect was associated with in situ downregulation of inflammatory mediators such as tumor necrosis factor alpha (TNF-alpha) and monocyte chemoattractant protein 1 (MCP-1), upregulation of transforming growth factor beta (TGF-beta), and preservation of BBB integrity. Our results indicate that intrathecal delivery of HSV-1-derived vectors containing antiinflammatory cytokine genes may play a major role in the future therapeutic armamentarium of inflammatory CNS-confined demyelinating diseases and, in particular, in the most fulminant forms where conventional therapeutic approaches have, so far, failed to achieve a satisfactory control of the disease evolution.


Subject(s)
Autoimmune Diseases/genetics , Central Nervous System/metabolism , Encephalomyelitis/genetics , Genetic Therapy/methods , Genetic Vectors , Herpesvirus 1, Human/genetics , Interleukin-4/genetics , Animals , Autoimmune Diseases/prevention & control , Blood-Brain Barrier , Brain/pathology , Cell Division , Chemokine CCL2/biosynthesis , Cytokines/biosynthesis , Down-Regulation , Encephalomyelitis/prevention & control , Enzyme-Linked Immunosorbent Assay , Humans , Interleukin-4/biosynthesis , Macaca mulatta , Magnetic Resonance Imaging , Male , Reverse Transcriptase Polymerase Chain Reaction , Spinal Cord/immunology , T-Lymphocytes/cytology , Time Factors , Transforming Growth Factor beta/biosynthesis , Tumor Necrosis Factor-alpha/biosynthesis , Up-Regulation
3.
J Neurol Neurosurg Psychiatry ; 67(6): 785-8, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10567499

ABSTRACT

The cysteine protease caspase-1 plays a crucial part in the inflammatory process due to its ability to proteolitically activate proinflammatory cytokine precursors, such as interleukin (IL)-1beta and IL-18. Multiple sclerosis is a chronic inflammatory demyelinating disease of the CNS in which the pathogenic process is mainly orchestrated by proinflammatory cytokines. The role of caspase-1 in multiple sclerosis was evaluated by measuring its mRNA levels in peripheral blood mononuclear cells (PBMCs) from seven patients with relapsing-remitting multiple sclerosis every 15 days over a 1 year period. The recorded levels were compared with clinical and MRI evidence of disease activity. Brain MRI was performed monthly in each patient. Caspase-1 mRNA levels were significantly increased in PBMCs from patients with multiple sclerosis compared with healthy controls (p<0.001). In patients with multiple sclerosis, a twofold to threefold increase of caspase-1 mRNA mean level was found in the week preceding an acute attack (p<0. 05). The magnitude of caspase-1 mRNA increase correlated with the number of new (p=0.01) but not persisting gadolinium enhancing brain MRI lesions. In conclusion, caspase-1 might be involved in the immune mediated process underlying CNS inflammation and might represent a suitable peripheral immunological marker of disease activity in multiple sclerosis.


Subject(s)
Brain/metabolism , Caspase 1/genetics , Caspase 1/metabolism , Leukocytes, Mononuclear/metabolism , Multiple Sclerosis/genetics , Multiple Sclerosis/metabolism , RNA, Messenger/genetics , Adult , Brain/pathology , DNA, Complementary/genetics , Female , Genetic Markers , Humans , Interleukin-1/genetics , Interleukin-1/metabolism , Interleukin-18/genetics , Interleukin-18/metabolism , Magnetic Resonance Imaging , Male , Protein Precursors/metabolism , Reverse Transcriptase Polymerase Chain Reaction/methods , Severity of Illness Index
4.
Eur Cytokine Netw ; 10(3): 431-6, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10477400

ABSTRACT

Demyelination is the main pathological feature of multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system. Tumor necrosis factor-alpha (TNF-alpha) can cause myelin damage and contribute to MS pathogenesis. We measured plasma and cerebrospinal fluid (CSF) levels of TNF-alpha and its soluble receptors, TNF-sRp55 and TNF-sRp75, in 18 patients with active MS, and in neurological and healthy controls. The same determinations were repeated on plasma and on CSF samples that were collected after the MS patients had ended a six-day treatment with high-dose methylprednisolone (MP). Pre- and post-treatment plasma and CSF TNF-alpha levels, when detectable, and those of TNF-sRp75, did not vary, and were similar to those of controls. CSF TNF-sRp55 levels were higher in acute MS patients than in controls. Post-treatment CSF TNF-sRp55 levels were higher than in the active phase of the disease. The MS patients, who clinically improved, tended to have the highest CSF TNF-sRp55 levels. The increase was due to intrathecal TNF-sRp55 synthesis. Although it is involved in MS pathogenesis, TNF-alpha is not detectable in plasma or in CSF samples from MS patients in various phases of the disease. A better marker of disease activity seems to be CSF TNF-sRp55 levels. The increased CSF levels of TNF-sRp55 in response to MP circumstantially suggest that this receptor could partially account for the beneficial effects of MP in acute MS.


Subject(s)
Methylprednisolone/therapeutic use , Multiple Sclerosis/blood , Multiple Sclerosis/cerebrospinal fluid , Receptors, Tumor Necrosis Factor/metabolism , Tumor Necrosis Factor-alpha/metabolism , Adult , Female , Humans , Male , Multiple Sclerosis/drug therapy , Receptors, Tumor Necrosis Factor/blood , Tumor Necrosis Factor-alpha/cerebrospinal fluid
5.
J Immunol ; 163(5): 2403-9, 1999 Sep 01.
Article in English | MEDLINE | ID: mdl-10452974

ABSTRACT

T cell-mediated inflammation is considered to play a key role in the pathogenic mechanisms sustaining multiple sclerosis (MS). Caspase-1, formerly designated IL-1beta-converting enzyme, is crucially involved in immune-mediated inflammation because of its pivotal role in regulating the cellular export of IL-1beta and IL-18. We studied the role of caspase-1 in experimental autoimmune encephalomyelitis (EAE), the animal model for MS. Caspase-1 is transcriptionally induced during EAE, and its levels correlate with the clinical course and transcription rate of proinflammatory cytokines such as TNF-alpha, IL-1beta, IFN-gamma, and IL-6. A reduction of EAE incidence and severity is observed in caspase-1-deficient mice, depending on the immunogenicity and on the amount of the encephalitogenic myelin oligodendrocyte glycoprotein (MOG) peptide used. In caspase-1-deficient mice, reduced EAE incidence correlates with defective development of anti-MOG IFN-gamma-producing Th1 cells. Finally, pharmacological blockade of caspase-1 in Biozzi AB/H mice, immunized with spinal cord homogenate or MOG35-55 peptide, by the caspase-1-inhibitor Z-Val-Ala-dl -Asp-fluoromethylketone, significantly reduces EAE incidence in a preventive but not in a therapeutic protocol. These results indicate that caspase-1 plays an important role in the early stage of the immune-mediated inflammatory process leading to EAE, thus representing a possible therapeutic target in the acute phase of relapsing remitting MS.


Subject(s)
Caspase 1/physiology , Encephalomyelitis, Autoimmune, Experimental/enzymology , Encephalomyelitis, Autoimmune, Experimental/immunology , Myelin Sheath/physiology , Spinal Cord/pathology , Spinal Cord/physiopathology , Amino Acid Chloromethyl Ketones/administration & dosage , Animals , Autoimmune Diseases/enzymology , Autoimmune Diseases/genetics , Autoimmune Diseases/pathology , Autoimmune Diseases/physiopathology , Caspase 1/deficiency , Caspase 1/genetics , Caspase Inhibitors , Cysteine Proteinase Inhibitors/administration & dosage , Disease Susceptibility , Encephalomyelitis, Autoimmune, Experimental/genetics , Encephalomyelitis, Autoimmune, Experimental/prevention & control , Female , Immunosuppressive Agents/administration & dosage , Infusion Pumps, Implantable , Mice , Mice, Inbred C57BL , Mice, Knockout , Myelin Sheath/pathology , Neuroprotective Agents/administration & dosage , RNA, Messenger/biosynthesis , Spinal Cord/immunology , Th1 Cells/enzymology , Th1 Cells/immunology , Up-Regulation/immunology
6.
Clin Chem ; 45(3): 400-5, 1999 Mar.
Article in English | MEDLINE | ID: mdl-10053042

ABSTRACT

BACKGROUND: Acetylcholine receptor (AChR) from human muscles is the antigen used currently in radioimmunoprecipitation assays (RIPAs) for the determination of anti-AChR antibodies in the diagnosis of myasthenia gravis (MG). Our aim was to develop and validate an ELISA using TE671 cells as the source of AChR. METHODS: After TE671 cell homogenization, the crude AChR extract was used for plate coating. Anti-AChR antibodies were determined in 207 MG patients and in 77 controls. RESULTS: The mean intra- and interassay CVs (for two samples with different anti-AChR antibody concentrations) were 9.7% and 15.7%, respectively. Test sensitivity and specificity, for generalized MG, were 79.5% (95% confidence interval, 72.8-85.0%) and 96.1% (89.0-99.1%). The detection limit was 2 nmol/L. Anti-AChR antibody concentrations from 53 MG patients, as tested with our ELISA, showed good agreement with an RIPA with a mean difference (SD) of 1.0 (5.6) nmol/L. CONCLUSION: Our ELISA is a simple screening test for the diagnosis of MG and enables rapid and inexpensive patient follow-up.


Subject(s)
Antibodies/blood , Myasthenia Gravis/immunology , Receptors, Cholinergic/immunology , Adolescent , Adult , Calibration , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , Male , Middle Aged , Myasthenia Gravis/blood , Reproducibility of Results , Sensitivity and Specificity , Tumor Cells, Cultured
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