Gene therapy for rheumatoid arthritis

Rheumatoid arthritis (RA) is a severe autoimmune systemic disease. Chronic synovial inflammation results in destruction of the joints. No conventional treatment is efficient in RA. Gene therapy of RA targets mainly the players of inflammation or articular destruction: TNF-alpha or IL-1 blocking agents (such as anti-TNF-alpha monoclonal antibodies, soluble TNF-alpha receptor, type II soluble receptor of IL-1, IL-1 receptor antagonist), antiinflammatory cytokines (such as IL-4, IL-10, IL-1), and growth factors. In this polyarticular disease, the vector expressing the therapeutic protein can be administered as a local (intra-articular injection) or a systemic treatment (extra-articular injection). All the main vectors have been used in experimental models, including the more recent lentivirus and adeno-associated virus. Ex vivo gene transfer was performed with synovial cells, fibroblasts, T cells, dendritic cells, and different cells from xenogeneic origin. In vivo gene therapy is simpler, although a less controlled method. Clinical trials in human RA have started with ex vivo retrovirus-expressing IL-1 receptor antagonists and have demonstrated the feasibility of the strategy of gene therapy. The best target remains to be determined and extensive research has to be conducted in preclinical studies.     

A novel in-vitro system for the simultaneous exposure of bladder smooth muscle cells to mechanical strain and sustained hydrostatic pressure

The novel hydrostrain system was designed in an effort to establish and maintain conditions that simulate the in-vivo mechanical environment of the bladder. In this laboratory system, ovine bladder smooth muscle cells on flexible, 10-cm-dia silastic membranes were exposed simultaneously to hydrostatic pressure (40 cm H2O, a pressure level currently associated with bladder pathologies) and mechanical strains (up to 25 percent) under standard cell culture conditions for 7 h. Under these conditions, Heparin Binding-Epidermal Growth Factor and Collagen Type III mRNA expression were significantly increased (p<0.01 and 0.1, respectively); however, no changes were observed in Collagen Type I mRNA expression. Decreases in the Collagen Type I:Type III ratio following simultaneous exposure of bladder smooth muscle cells to pathological levels of hydrostatic pressure and mechanical strain in vitro are in agreement with clinically observed increases in Collagen Type III with concomitant decreased human bladder compliance. The results of the present study, therefore, provide cellular/molecular level information relevant to bladder pathology that could have significant implications in the field of clinical urology.     

Structural investigation of the ligand binding domain of the zebrafish VDR in complexes with 1alpha,25(OH)2D3 and Gemini: purification, crystallization and preliminary X-ray diffraction analysis

The nuclear receptor of Vitamin D can be activated by a large number of agonist molecules with a wide spectrum in their stereochemical framework. Up to now most of our structural information related to the protein-ligand complex formation is based on an engineered ligand binding domain (LBD) of the human receptor. We now have extended our database, using a wild-type LBD from zebrafish that confirms the previously reported results and allows to investigate the binding of ligands that induce significant conformational changes at the protein level.     

Flow-conditioned HUVECs support clustered leukocyte adhesion by coexpressing ICAM-1 and E-selectin

Endothelial sequestration of circulating monocytes is a key event in early atherosclerosis. Hemodynamics is proposed to regulate monocyte-endothelial cell interactions by direct cell activation and establishment of flow environments that are conducive or prohibitive to cell-cell interaction. We investigated fluid shear regulation of monocyte-endothelial cell adhesion in vitro using a disturbed laminar shear system that models in vivo hemodynamics characteristic of lesion-prone vascular regions. Human endothelial cell monolayers were flow conditioned for 6 h before evaluation of monocyte adhesion under static and dynamic flow conditions. Results revealed a distinctive clustered cell pattern of monocyte adhesion that strongly resembles in vivo leukocyte adhesion in early- and late-stage atherosclerosis. Clustered monocyte cell adhesion correlated with endothelial cells coexpressing intercellular adhesion molecule-1 (ICAM-1) and E-selectin as result of a flow-induced, selective upregulation of E-selectin expression in a subset of ICAM-1-expressing cells. Clustered monocyte cell adhesion assayed under static conditions exhibited a spatial variation in size and frequency of occurrence, which demonstrates differential regulation of endothelial cell adhesiveness by the local flow environment. Dynamic adhesion studies conducted with circulating monocytes resulted in clustered cell adhesion only within the disturbed flow region, where the monocyte rate of motion is sufficiently low for cell-cell interaction. These studies provide evidence and reveal mechanisms of local hemodynamic regulation of endothelial adhesiveness and endothelial monocyte interaction that lead to localized monocyte adhesion and potentially contribute to the focal origin of arterial diseases such as atherosclerosis.     

Understanding the Anodic Mechanism of a Seafloor Fuel Cell: Interactions Between Geochemistry and Microbial Activity

Seafloor fuel cells made with graphite electrodes generate electricity by promoting electron transfer in response to a natural voltage difference (−0.7 to −0.8 V) between anoxic sediments and overlying oxic seawater. Geochemical impacts of a seafloor fuel cell on sediment solids and porewaters were examined to identify the anodic mechanisms and substrates available for current production. In an estuarine environment with little dissolved sulfide, solid-phase acid volatile sulfide and Cr²⁺-reducible sulfur minerals decreased significantly toward the anode after 7 months of nearly continuous energy harvesting. Porewater iron and sulfate increased by millimolar amounts. Scanning electron microscope images showed a biofilm overcoating the anode, and electron microprobe analyses revealed accumulations of sulfur, iron, silicon and phosphorus at the electrode surface. Sulfur deposition was also observed on a laboratory fuel cell anode used to generate electricity with only dissolved sulfide as an electron donor. Moreover, current densities and voltages displayed by these purely chemical cells were similar to the values measured with field devices. These results indicate that electron transfer to seafloor fuel cells can readily result in the oxidation of dissolved and solid-phase forms of reduced sulfur producing mainly S⁰ which deposits at the electrode surface. This oxidation product is consistent with the observed enrichment of bacteria most closely related to Desulfobulbus/Desulfocapsa genera within the anode biofilm, and its presence is proposed to promote a localized biogeochemical cycle whereby biofilm bacteria regenerate sulfate and sulfide. This electron-shuttling mechanism may co-occur while these or other bacteria use the anode directly as a terminal electron acceptor.     

Maternal smoking is associated with mitochondrial DNA depletion and respiratory chain complex III deficiency in placenta

Maternal smoking during pregnancy is often associated with a decrease in placental function, which might lead to intrauterine growth retardation. Because tobacco is known to alter the mitochondrial respiratory function in cardiomyocytes and lung tissue, we hypothesized that placental mitochondrial function could be altered by maternal smoking. Placental mitochondria from 9 smoking and 19 nonsmoking mothers were isolated by differential centrifugation. Mitochondrial oxygen consumption was measured by polarography, and the enzymatic activity of each complex of the electron transport chain was assessed by spectrophotometry. In addition, the relative content in mitochondrial DNA (mtDNA) was determined by real-time quantitative PCR in placentas from seven smoking and seven nonsmoking mothers. We observed a 29% reduction in the enzymatic activity of complex III in the placental mitochondria from smokers compared with nonsmokers (P = 0.03). The relative content of mtDNA (with respect to the beta-globin gene) was reduced by 37% in the placental tissue from smokers compared with nonsmokers (P < 0.02). Both the enzymatic activity of complex III and mtDNA content were inversely related with the daily consumption of cigarettes, and mtDNA content was correlated with cord blood insulin-like growth factor-binding protein-3 (r = 0.74, P < 0.01), a marker of fetal growth. These results show that maternal smoking is associated with placental mitochondrial dysfunction, which might contribute to restricted fetal growth by limiting energy availability in cells.     

Differential effects of shear stress and cyclic stretch on focal adhesion remodeling, site-specific FAK phosphorylation, and small GTPases in human lung endothelial cells

Regulation of endothelial cell (EC) permeability by bioactive molecules is associated with specific patterns of cytoskeletal and cell contact remodeling. A role for mechanical factors such as shear stress (SS) and cyclic stretch (CS) in cytoskeletal rearrangements and regulation of EC permeability becomes increasingly recognized. This paper examined redistribution of focal adhesion (FA) proteins, site-specific focal adhesion kinase (FAK) phosphorylation, small GTPase activation and barrier regulation in human pulmonary EC exposed to laminar shear stress (15 dyn/cm2) or cyclic stretch (18% elongation) in vitro. SS caused peripheral accumulation of FAs, whereas CS induced randomly distributed FAs attached to the ends of newly formed stress fibers. SS activated small GTPase Rac without effects on Rho, whereas 18% CS activated without effect on Rac. SS increased transendothelial electrical resistance (TER) in EC monolayers, which was further elevated by barrier-protective phospholipid sphingosine 1-phosphate. Finally, SS induced FAK phosphorylation at Y576, whereas CS induced FAK phosphorylation at Y397 and Y576. These results demonstrate for the first time differential effects of SS and CS on Rho and Rac activation, FA redistribution, site-specific FAK phosphorylation, and link them with SS-mediated barrier enhancement. Thus, our results suggest common signaling and cytoskeletal mechanisms shared by mechanical and chemical factors involved in EC barrier regulation.     

Integrative Model of the Immune Response to a Pulmonary Macrophage Infection: What Determines the Infection Duration?

The immune mechanisms which determine the infection duration induced by pathogens targeting pulmonary macrophages are poorly known. To explore the impact of such pathogens, it is indispensable to integrate the various immune mechanisms and to take into account the variability in pathogen virulence and host susceptibility. In this context, mathematical models complement experimentation and are powerful tools to represent and explore the complex mechanisms involved in the infection and immune dynamics. We developed an original mathematical model in which we detailed the interactions between the macrophages and the pathogen, the orientation of the adaptive response and the cytokine regulations. We applied our model to the Porcine Respiratory and Reproductive Syndrome virus (PRRSv), a major concern for the swine industry. We extracted value ranges for the model parameters from modelling and experimental studies on respiratory pathogens. We identified the most influential parameters through a sensitivity analysis. We defined a parameter set, the reference scenario, resulting in a realistic and representative immune response to PRRSv infection. We then defined scenarios corresponding to graduated levels of strain virulence and host susceptibility around the reference scenario. We observed that high levels of antiviral cytokines and a dominant cellular response were associated with either short, the usual assumption, or long infection durations, depending on the immune mechanisms involved. To identify these mechanisms, we need to combine the levels of antiviral cytokines, including , and . The latter is a good indicator of the infected macrophage level, both combined provide the adaptive response orientation. Available PRRSv vaccines lack efficiency. By integrating the main interactions between the complex immune mechanisms, this modelling framework could be used to help designing more efficient vaccination strategies.