Electrotransfer of CpG free plasmids enhances gene expression in skin.

Skin is a very suitable target for gene therapy and DNA vaccination due to its accessibility, its surface and its ability to produce transgenes. Gene electrotransfer (GET) to the skin is under development for clinical applications for DNA vaccine or local treatment such as wound healing. Local treatments are effective if the expression of the plasmid affects only the local environment (skin) by inducing an efficient concentration over a prolonged period. In this study, we evaluate the control of expression in the skin of a plasmid coding a fluorescent protein by its CpG (cytosine-phosphate-guanine motif) content. Two fluorescent reporter genes are evaluated: tdTomato and GFP. The expression is followed on the long term by in vivo fluorescence imaging. Our results show that GET mediated expression in the skin can be controlled by the CpG content of the plasmid. Long term expression (>120 days) can be obtained at high level with CpG-free constructs associated with a proper design of the electrodes where the field distribution mediating the gene electrotransfer is present deep in the skin.

Minicircle DNA electrotransfer for efficient tissue-targeted gene delivery.

A major issue for successful human gene therapy or genetic vaccination is a safe high-transgene expression level. Plasmid-based (non-viral) physical methods of gene transfer offered attracting approaches but their low efficiencies have limited their use in human pre-clinical trials. One of the limits appears to be the size of the plasmid that must be transferred across the cell membrane to the nucleus for its processing. In the present work to enhance gene transfer and expression, we evaluated a new generation of DNA vector; the minicircle, combined with the electropulsation technique. Minicircle is a doubled-stranded circular DNA with reduced size as it is devoid of bacterial sequences. We showed that electrotransferred minicircle encoding green fluorescent protein had higher in vitro transfection level compared with full-length plasmid. We demonstrated that minicircle great efficiency was not because of cellular toxicity decrease but was correlated to more efficient vector uptake by cells. Vector electrotransfection was operated in vivo and, using fluorescence imaging, minicircle electrotransfer was shown to enhance the efficiency and duration of tissue-targeted gene delivery and expression. By combining powerful expression and delivery systems, we have provided a valuable method for new approaches in gene therapy and genetic vaccination.

Drug delivery by electropulsation: Recent developments in oncology.

Electro-permeabilisation allows the free access of polar compounds to the cytoplasm by a reversible alteration of the cell membrane. It is now used in clinics for the eradication of cutaneous solid tumors. New developments predict its future applications for other anti-cancer treatments.

Cholera toxin, E. coli heat-labile toxin, and non-toxic derivatives induce dendritic cell migration into the follicle-associated epithelium of Peyer's patches.

The follicle-associated epithelium (FAE) of Peyer's patches (PPs) transports antigens and microorganisms into mucosal lymphoid tissues where they are captured by subepithelial dendritic cells (DCs). Feeding of cholera toxin (CT) induced migration of subepithelial DCs to interfollicular T-cell areas within 24 h. This study investigated short-term effects of CT, Escherichia coli heat-labile toxin, and non-toxic derivatives on DC migration. CT or CTB injected into ligated intestinal loops induced significant increase in CD11c+ DCs within the FAE within 90 min. In mice fed CT intragastrically, DC numbers in the FAE increased by 1 h, were maximal by 2 h, declined between 8 and 12 h, and were reversed by 24 h. Feeding of native LT, recombinant CTB, dibutyryl cyclic AMP, and to a lesser extent mutated CT(E29H) or mutated LT(R192G) had the same effect. Thus, both A and B subunits of enterotoxins, presumably acting through distinct signaling pathways, may promote capture of incoming antigens and pathogens by PP DCs.

Heterologous prime-boost strategy to immunize very young infants against measles: pre-clinical studies in rhesus macaques.

Infants in developing countries are at high risk of developing severe clinical measles if they become infected during the "window of vulnerability" (age 4-9 months), when declining maternal antibodies do not protect against wild virus, yet impede successful immunization by attenuated measles vaccine. We developed two Sindbis replicon-based DNA vaccines expressing measles virus hemagglutinin and fusion protein with the goal of priming young infants to respond safely and effectively to subsequent boosting with attenuated measles vaccine. Intradermal prime with DNA vaccines by needle-free injection followed by aerosol or parenteral boost with licensed measles vaccine was well tolerated by juvenile and young infant rhesus macaques, and protected against clinical measles and viremia on wild-type virus challenge. A proteosome-measles vaccine administered alone (three doses) or as a boost following DNA vaccine priming was also safe and protective. These promising results pave the way for clinical trials to assess this prime-boost strategy.

Cytokine production in T lymphocyte-microglia interaction is attenuated by glatiramer acetate: a mechanism for therapeutic efficacy in multiple sclerosis.

The efficacy of glatiramer acetate in multiple sclerosis (MS) is thought to involve the production of Th2 regulatory lymphocytes that secrete anti-inflammatory cytokines; however, other mechanisms cannot be excluded Given that activated T lymphocytes infiltrate into the CNS and become in dose proximity to microglia, we evaluated whether glatiramer acetate affects the potential interaction between T cells and microglia. We report that the co-culture of activated T lymphocytes with microglia led to the induction of several cytokines, and that these were reduced by glatiramer acetate treatment Morphological transformation of bipolar/ramified microglia into an activated ameboid form was attenuated by glatiramer acetate. These results reveal a novel mechanism for glatiramer acetate: the impairment of activated T cells to effectively interact with microglia to produce cytokines. The net result of a non-inflammatory milieu within the CNS, in spite of T cell infiltration, may help account for the amelioration of disease activity in MS patients on glatiramer acetate therapy.

Fatal adenovirus type 7b infection in a child with Smith-Lemli-Opitz syndrome.

Adenovirus type 7 causes worldwide respiratory tract infections, mainly in children. Severe systemic infections can occur, especially in immunocompromised patients and in patients with underlying chronic diseases. This report describes the first case of a fatal disseminated adenovirus type 7 infection in a child with Smith-Lemli-Opitz syndrome, a rare autosomal recessive disorder due to a primary enzymatic defect in cholesterol metabolism. Nasopharyngeal secretions and autopsy specimens including liver, lung, pleural fluid, and rectum were collected for viral culture. Adenovirus serotype 7 strains were obtained from all anatomic sites, except the liver. All these clinical isolates were analyzed using restriction endonuclease digestion of the genome, identifying them as genome type 7b, a virulent type. In this case, the fatal evolution could have been accelerated by the presence of an immunodeficiency although immunodeficiency is not included in the definition of Smith-Lemli-Opitz syndrome. The frequent recurrent banal infections in Smith-Lemli-Opitz syndrome could be prevented by a cholesterol supplementation regimen. Finally, this report emphasizes the need for efficient therapy for disseminated adenovirus infections, especially for virulent genome types.

Cytokine production consequent to T cell--microglia interaction: the PMA/IFN gamma-treated U937 cells display similarities to human microglia.

Cognate interactions between human adult microglia and activated T lymphocytes induce the production of inflammatory cytokines. Since this interaction can occur in a non-antigen-dependent manner, it is relevant to a variety of CNS diseases where activated T cells, regardless of specificities, come into contact with microglia; these disorders include multiple sclerosis, trauma, stroke and Alzheimer's disease. A model cell line would facilitate studies of the engagement between T cells and human adult microglia, since the latter are difficult to obtain in substantial quantity or frequency. This study shows that the PMA/IFN gamma-treated U937 cell line shows similarities to microglia in its interaction with activated T lymphocytes, in that the production of tumor necrosis factor (TNF)-alpha, interleukin (IL)-4, IL-10 and IL-12 is induced. Morphological features and mechanisms of cytokine production resemble those observed in microglia--T cell co-cultures since CTLA-4 and CD40--CD40L blockades reduce TNF-alpha and IL-10 levels, while anti-CD23 inhibits IL-10 only in U937--T cell interactions. We propose that PMA/IFN gamma-treated U937 cells can serve as a model of human adult microglia to study cytokine generation in response to interactions with activated T cells.

Interferon beta-1b increases interleukin-10 in a model of T cell-microglia interaction: relevance to MS.

BACKGROUND: The modes of action of interferon beta (IFN-beta) in MS remain unclear, but enhanced levels of the anti-inflammatory cytokine interleukin-10 (IL-10) in the CSF of patients with MS may be a marker of its prognostic efficacy. OBJECTIVE: To examine potential mechanisms by which IL-10 may be increased by IFN-ss in the milieu of the CNS. METHODS: A model of T cell interaction with microglia in vitro was used. Production of cytokines was monitored by measuring the levels of various cytokine proteins, using ELISA. RESULTS: Pretreatment of T cells with IFN-beta potentiates the production of IL-10 when they interact with adult human microglia, human fetal microglia, or U937 cells treated with phorbol-12-myristate-13-acetate (PMA) and IFN-gamma. The enhancing effect of IFN-beta on IL-10 requires cell-cell contact, but does not seem to depend on pathways implicated in microglia-T cell interactions, involving CD40, CD23, and B7. In contrast to IL-10, IFN-beta inhibits the production of other cytokines, including tumor necrosis factor-alpha (TNF-alpha), IL-1beta, IL-4, IL-12, and IL-13. CONCLUSIONS: The increase of IL-10 in microglia-T cell interaction by IFN-beta together with a decrease of other cytokines may lead to a noninflammatory milieu in the CNS. This mechanism could contribute to the efficacy of IFN-beta in MS.

Mechanisms of IL-10 production in human microglia-T cell interaction.

IL-10, a cytokine with important anti-inflammatory properties, is generated within the CNS during neuroinflammation. The mechanism for its production is poorly understood. Since infiltrating lymphocytes come into close proximity with the macrophage-like cells of the CNS, the microglia, we have used an in vitro human microglia-T cell coculture system to address the mechanisms of IL-10 production. We demonstrate that microglia or activated T cells alone secrete negligible amounts of IL-10, but that their coculture results in significant IL-10 production, which was effected by both cell types. IL-10 generation was cell contact dependent, and treatment with anti-CD40, CTLA-4-Fc, or anti-CD23 decreased the IL-10 content in microglia-T cell cocultures. The combination of anti-CD40 and CTLA-4-Fc reduced IL-10 levels to the negligible amounts seen with T cells or microglia in isolation. By also measuring TNF-alpha levels, specificity of cytokine regulation was observed; while anti-CD40 and CTLA-4-Fc reduced IL-10 and TNF-alpha levels, anti-CD23 did not affect TNF-alpha while attenuating IL-10 generation. Anti-very late Ag-4, which decreased TNF-alpha levels, did not affect IL-10. These results implicate the CD40, B7, and CD23 pathways in IL-10 production following microglia-T cell encounter and have relevance to the regulation of an anti-inflammatory response within the CNS.

Interferon beta in the treatment of multiple sclerosis: mechanisms of action.

Interferon beta (IFN-beta) has been shown in several clinical trials to have efficacy in MS. Its mechanism of action, however, remains unclear. In this review, several biological activities of IFN-beta are highlighted, including its inhibitory effects on proliferation of leukocytes and antigen presentation. Furthermore, IFN-beta may modulate the profile of cytokine production toward that of the anti-inflammatory phenotype, and this appears to occur in the systemic circulation and within the CNS. Finally, IFN-beta can reduce T-cell migration by inhibiting the activity of T-cell matrix metalloproteinases. These activities are likely to act in concert to account for the mechanism of IFN-beta in MS.

Chemokine-enhanced migration of human peripheral blood mononuclear cells is antagonized by interferon beta-1b through an effect on matrix metalloproteinase-9.

The increased migration of peripheral blood mononuclear cells (PBMNCs) across a fibronectin (FN) matrix in response to the chemokines RANTES, MIP-1 alpha and MCP-1 is antagonized by interferon-beta-1b (IFN beta-1b). MCP-1 treatment of PBMNCs elevates their mRNA level and secretion of a matrix degrading enzyme, matrix metalloproteinase (MMP)-9, which is abrogated by IFN beta-1b. The clinical benefits of IFN beta-1b treatment in multiple sclerosis patients may in part be a result of this drug's ability to decrease the migration of PBMNCs in spite of a chemotactic gradient. Furthermore, the elevation of MMP-9 production by PBMNCs may be an important mechanism of action of chemokines.

Microglial production of TNF-alpha is induced by activated T lymphocytes. Involvement of VLA-4 and inhibition by interferonbeta-1b.

TNF-alpha is a proinflammatory cytokine involved in many inflammatory conditions such as Crohn's disease, rheumatoid arthritis, cachexia, AIDS, and multiple sclerosis (MS). TNF-alpha is produced mainly by cells of the macrophage lineage, which includes microglia in the central nervous system. Here, we describe a mechanism through which TNF-alpha is generated by microglia. We show that activated human T lymphocytes induce the microglial production of TNF-alpha, and that is attenuated by a functional blocking antibody to CD49d, the alpha chain of the VLA-4 integrin on T cells. We also report that interferonbeta-1b (IFNbeta-1b), a drug that alleviates symptoms in MS, downregulates the expression of CD49d and reduces TNF-alpha production, mechanisms which can help account for its efficacy in MS.

Astrogliosis in the neonatal and adult murine brain post-trauma: elevation of inflammatory cytokines and the lack of requirement for endogenous interferon-gamma.

The relevance of astrogliosis remains controversial, especially with respect to the beneficial or detrimental influence of reactive astrocytes on CNS recovery. This dichotomy can be resolved if the mediators of astrogliosis are identified. We have measured the levels of transcripts encoding inflammatory cytokines in injury systems in which the presence or absence of astrogliosis could be produced selectively. A stab injury to the adult mouse brain using a piece of nitrocellulose (NC) membrane elicited a prompt and marked increase in levels of transcripts for interleukin (IL)-1alpha, IL-1beta, and tumor necrosis factor (TNF)-alpha, which are considered to be microglia/macrophage cytokines. The elevations preceded, or occurred concomitantly with, the rise in glial fibrillary acidic protein mRNA, an early manifestation of astrogliosis. In neonatal mice, IL-1 and TNF-alpha mRNA were elevated to a greater extent by an NC-implant injury, which produced astrogliosis, than after an NC-stab, with minimal astrogliosis. We determined whether endogenous interferon (IFN)-gamma could be responsible for the observed increases in IL-1 and TNF-alpha, because IFN-gamma is a potent microglia/macrophage activator, and because its exogenous administration to rodents enhanced astrogliosis after adult or neonatal insults. A lack of requirement for endogenous IFN-gamma was demonstrated by three lines of evidence. First, no increase in IFN-gamma transcripts could be found at injury. Second, the administration of a neutralizing antibody to IFN-gamma did not attenuate astrogliosis. Third, in IFN-gamma knockout adult mice, astrogliosis and increases in levels of IL-1alpha and TNF-alpha were induced rapidly by injury. The marked elevation of inflammatory cytokines is discussed in the context of astrogliosis and general CNS recovery.

Hyphal growth promotion in vitro of the VA mycorrhizal fungus, Gigaspora margarita Becker & Hall, by the activity of structurally specific flavonoid compounds under CO2 -enriched conditions.

Plant phenolic compounds are known to be inducers of virulence genes in plant-pathogen interactions such as those involving Agrobacterium, and flavonoids are known to be inducers or inhibitors of Nod genes in Rhizobium-legume symbiosis. More recent studies suggest that some of these compounds act as molecular signals in the development of vesicular-arbuscular mycorrhizas (VAM). The present study has shown that hyphal growth of the VAM fungus, Gigaspora margarita Becker & Hall, is affected by both stimulatory and inhibitory flavonoids, when applied at 10 µ together with an optimal carbon dioxide enrichment. Stimulatory compounds were all flavonols (kaempferol, quercetin and morin) and possessed at least one hydroxyl group on the B ring. Conversely, two isoflavones (biochanin A, and genistein), a single flavanone (hesperetin) and two compounds without any hydroxyl group on the B ring, galangin (flavonol) and chrysin (flavone), were all inhibitors of hyphal growth.

Panic disorder in cardiac outpatients.

The prevalence of and nature of panic disorder were investigated in an ambulatory cardiology practice. Questionnaires about panic symptoms were mailed to 414 patients, and possible or definite panic disorder was found in 104 of the 310 respondents. Interviews with 52 of the 104 patients substantiated diagnoses of panic disorder, for a prevalence of 9.2% of the total sample population of 414. Comparison of patients grouped by duration of panic disorder revealed that long-duration panic disorder had its onset before age 30 and followed a chronic course. Short-duration panic disorder developed at an older age following the appearance of cardiac disease.