An Adeno-Associated Virus-Based Intracellular Sensor of Pathological Nuclear Factor-κB Activation for Disease-Inducible Gene Transfer.

Stimulation of resident cells by NF-κB activating cytokines is a central element of inflammatory and degenerative disorders of the central nervous system (CNS). This disease-mediated NF-κB activation could be used to drive transgene expression selectively in affected cells, using adeno-associated virus (AAV)-mediated gene transfer. We have constructed a series of AAV vectors expressing GFP under the control of different promoters including NF-κB -responsive elements. As an initial screen, the vectors were tested in vitro in HEK-293T cells treated with TNF-α. The best profile of GFP induction was obtained with a promoter containing two blocks of four NF-κB -responsive sequences from the human JCV neurotropic polyoma virus promoter, fused to a new tight minimal CMV promoter, optimally distant from each other. A therapeutical gene, glial cell line-derived neurotrophic factor (GDNF) cDNA under the control of serotype 1-encapsidated NF-κB -responsive AAV vector (AAV-NF) was protective in senescent cultures of mouse cortical neurons. AAV-NF was then evaluated in vivo in the kainic acid (KA)-induced status epilepticus rat model for temporal lobe epilepsy, a major neurological disorder with a central pathophysiological role for NF-κB activation. We demonstrate that AAV-NF, injected in the hippocampus, responded to disease induction by mediating GFP expression, preferentially in CA1 and CA3 neurons and astrocytes, specifically in regions where inflammatory markers were also induced. Altogether, these data demonstrate the feasibility to use disease-activated transcription factor-responsive elements in order to drive transgene expression specifically in affected cells in inflammatory CNS disorders using AAV-mediated gene transfer.

Peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) but not PPARalpha serves as a plasma free fatty acid sensor in liver.

Peroxisome proliferator-activated receptor alpha (PPARalpha) is an important transcription factor in liver that can be activated physiologically by fasting or pharmacologically by using high-affinity synthetic agonists. Here we initially set out to elucidate the similarities in gene induction between Wy14643 and fasting. Numerous genes were commonly regulated in liver between the two treatments, including many classical PPARalpha target genes, such as Aldh3a2 and Cpt2. Remarkably, several genes induced by Wy14643 were upregulated by fasting independently of PPARalpha, including Lpin2 and St3gal5, suggesting involvement of another transcription factor. Using chromatin immunoprecipitation, Lpin2 and St3gal5 were shown to be direct targets of PPARbeta/delta during fasting, whereas Aldh3a2 and Cpt2 were exclusive targets of PPARalpha. Binding of PPARbeta/delta to the Lpin2 and St3gal5 genes followed the plasma free fatty acid (FFA) concentration, consistent with activation of PPARbeta/delta by plasma FFAs. Subsequent experiments using transgenic and knockout mice for Angptl4, a potent stimulant of adipose tissue lipolysis, confirmed the stimulatory effect of plasma FFAs on Lpin2 and St3gal5 expression levels via PPARbeta/delta. In contrast, the data did not support activation of PPARalpha by plasma FFAs. The results identify Lpin2 and St3gal5 as novel PPARbeta/delta target genes and show that upregulation of gene expression by PPARbeta/delta is sensitive to plasma FFA levels. In contrast, this is not the case for PPARalpha, revealing a novel mechanism for functional differentiation between PPARs.

The unfolded protein response: integrating stress signals through the stress sensor IRE1α.

Stress induced by accumulation of unfolded proteins at the endoplasmic reticulum (ER) is a classic feature of secretory cells and is observed in many tissues in human diseases including cancer, diabetes, obesity, and neurodegeneration. Cellular adaptation to ER stress is achieved by the activation of the unfolded protein response (UPR), an integrated signal transduction pathway that transmits information about the protein folding status at the ER to the nucleus and cytosol to restore ER homeostasis. Inositol-requiring transmembrane kinase/endonuclease-1 (IRE1α), the most conserved UPR stress sensor, functions as an endoribonuclease that processes the mRNA of the transcription factor X-box binding protein-1 (XBP1). IRE1α signaling is a highly regulated process, controlled by the formation of a dynamic scaffold onto which many regulatory components assemble, here referred to as the UPRosome. Here we provide an overview of the signaling and regulatory mechanisms underlying IRE1α function and discuss the emerging role of the UPR in adaptation to protein folding stress in specialized secretory cells and in pathological conditions associated with alterations in ER homeostasis.

Mismatch of arterial and central venous blood gas analysis during haemorrhage.

BACKGROUND AND OBJECTIVE: Arterial base excess and lactate levels are key parameters in the assessment of critically ill patients. The use of venous blood gas analysis may be of clinical interest when no arterial blood is available initially. METHODS: Twenty-four pigs underwent progressive normovolaemic haemodilution and subsequent progressive haemorrhage until the death of the animal. Base excess and lactate levels were determined from arterial and central venous blood after each step. In addition, base excess was calculated by the Van Slyke equation modified by Zander (BE(z)). Continuous variables were summarized as mean +/- SD and represent all measurements (n = 195). RESULTS: Base excess according to National Committee for Clinical Laboratory Standards for arterial blood was 2.27 +/- 4.12 versus 2.48 +/- 4.33 mmol(-l) for central venous blood (P = 0.099) with a strong correlation (r(2) = 0.960, P < 0.001). Standard deviation of the differences between these parameters (SD-DIFBE) did not increase (P = 0.355) during haemorrhage as compared with haemodilution. Arterial lactate was 2.66 +/- 3.23 versus 2.71 +/- 2.80 mmol(-l) in central venous blood (P = 0.330) with a strong correlation (r(2) = 0.983, P < 0.001). SD-DIFLAC increased (P < 0.001) during haemorrhage. BE(z) for central venous blood was 2.22 +/- 4.62 mmol(-l) (P = 0.006 versus arterial base excess according to National Committee for Clinical Laboratory Standards) with strong correlation (r(2) = 0.942, P < 0.001). SD-DIFBE(z)/base excess increased (P < 0.024) during haemorrhage. CONCLUSION: Central venous blood gas analysis is a good predictor for base excess and lactate in arterial blood in steady-state conditions. However, the variation between arterial and central venous lactate increases during haemorrhage. The modification of the Van Slyke equation by Zander did not improve the agreement between central venous and arterial base excess.

Electrochemical As(III) whole-cell based biochip sensor.

The development of a whole-cell based sensor for arsenite detection coupling biological engineering and electrochemical techniques is presented. This strategy takes advantage of the natural Escherichia coli resistance mechanism against toxic arsenic species, such as arsenite, which consists of the selective intracellular recognition of arsenite and its pumping out from the cell. A whole-cell based biosensor can be produced by coupling the intracellular recognition of arsenite to the generation of an electrochemical signal. Hereto, E. coli was equipped with a genetic circuit in which synthesis of beta-galactosidase is under control of the arsenite-derepressable arsR-promoter. The E. coli reporter strain was filled in a microchip containing 16 independent electrochemical cells (i.e. two-electrode cell), which was then employed for analysis of tap and groundwater samples. The developed arsenic-sensitive electrochemical biochip is easy to use and outperforms state-of-the-art bacterial bioreporters assays specifically in its simplicity and response time, while keeping a very good limit of detection in tap water, i.e. 0.8ppb. Additionally, a very good linear response in the ranges of concentration tested (0.94ppb to 3.75ppb, R(2)=0.9975 and 3.75 ppb to 30ppb, R(2)=0.9991) was obtained, complying perfectly with the acceptable arsenic concentration limits defined by the World Health Organization for drinking water samples (i.e. 10ppb). Therefore, the proposed assay provides a very good alternative for the portable quantification of As (III) in water as corroborated by the analysis of natural groundwater samples from Swiss mountains, which showed a very good agreement with the results obtained by atomic absorption spectroscopy.

Gas-filled microbubble-mediated delivery of antigen and the induction of immune responses.

The use of well characterized recombinant or purified protein antigens (Ag) for vaccination is of interest for safety reasons and in the case where inactivated pathogens are not available (cancer, allergy). However it requires the addition of adjuvants such as Ag carrier or immune stimulators to potentiate their immunogenicity. In this study, we demonstrated that gas-filled microbubbles (MB) can serve as an efficient Ag delivery system to promote phagocytosis of the model Ag ovalbumin (OVA) without the need of ultrasound application. Once internalized by DC, OVA was processed and presented to both CD4 and CD8 T cells in vitro; such observations were coupled with the capacity of MB to activate DC. In vivo administration of MB-associated OVA in naïve wild-type Balb/c mice resulted in the induction of OVA-specific antibody and T cell responses. Detailed characterization of the generated immune response demonstrated the production of both IgG1 and IgG2a serum antibodies, as well as the secretion of IFN-γ and IL-10 by splenocytes. Interestingly, similar results were obtained with human DC in regards of Ag delivery and cell activation. Therefore, the data presented here settle the proof of principle for the further evaluation of MB-based immunomodulation studies.

The phagocytosis of gas-filled microbubbles by human and murine antigen-presenting cells.

This study was designed to evaluate the potential of gas-filled microbubbles (MB) to be internalized by antigen-presenting cells (APC). Fluorescently labeled MB were prepared, thus permitting to track binding to, and internalization in, APC. Both human and mouse cells, including monocytes and dendritic cells (DC), prove capable to phagocyte MB in vitro. Observation by confocal laser scanning microscopy showed that interaction between MB and target cells resulted in a rapid internalization in cellular compartments and to a lesser extent in the cytoplasm. Capture of MB by APC resulted in phagolysosomal targeting as verified by double staining with anti-lysosome-associated membrane protein-1 monoclonal antibody and decrease of internalization by phagocytosis inhibitors. Fluorescent MB injected subcutaneously (s.c.) in mice were found to be associated with CD11c(+)DC in lymph nodes draining the injection sites 24 h after administration. Altogether, our study demonstrates that MB can successfully target APC both in vitro and in vivo, and thus may serve as a potent Ag delivery system without requirement for ultrasound-based sonoporation. This adds to the potential of applications of MB already extensively used for diagnostic imaging in humans.

Risk evaluation for spoofing against a sensor supplied with liveness detection

The aim of this paper is to evaluate the risks associated with the use of fake fingerprints on a livescan supplied with a method of liveness detection. The method is based on optical properties of the skin. The sensor uses several polarizations and illuminations to capture the information of the different layers of the human skin. These experiments also allow for the determination under which conditions the system is deceived and if there is an influence respectively of the nature of the fake, the mould used for the production or the individuals involved in the attack. These experiments showed that current multispectral sensors can be deceived by the use of fake fingerprints created with or without the cooperation of the subject. Fakes created from direct casts perform better than those produced by fakes created from indirect casts. The results showed that the success of the attack is influenced by two main factors. The first is the quality of the fakes, and by extension the quality of the original fingerprint. The second is the combination of the general patterns involved in the attacks since an appropriate combination can strongly increase the rates of successful attacks.

The endoplasmic reticulum: a sensor of cellular stress that modulates immune responses.

Many inflammatory and infectious diseases are characterized by the activation of signaling pathways steaming from the endoplasmic reticulum (ER). These pathways, primarily associated with loss of ER homeostasis, are emerging as key regulators of inflammation and infection. Recent advances shed light on the mechanisms linking ER-stress and immune responses.

Polyimide/SU-8 catheter-tip MEMS gauge pressure sensor.

This paper describes the development of a polyimide/SU-8 catheter-tip MEMS gauge pressure sensor. Finite element analysis was used to investigate critical parameters, impacting on the device design and sensing characteristics. The sensing element of the device was fabricated by polyimide-based micromachining on a flexible membrane, using embedded thin-film metallic wires as piezoresistive elements. A chamber containing this flexible membrane was sealed using an adapted SU-8 bonding technique. The device was evaluated experimentally and its overall performance compared with a commercial silicon-based pressure sensor. Furthermore, the device use was demonstrated by measuring blood pressure and heart rate in vivo.

Portal glucose infusion in the mouse induces hypoglycemia: evidence that the hepatoportal glucose sensor stimulates glucose utilization.

To analyze the role of the murine hepatoportal glucose sensor in the control of whole-body glucose metabolism, we infused glucose at a rate corresponding to the endogenous glucose production rate through the portal vein of conscious mice (Po-mice) that were fasted for 6 h. Mice infused with glucose at the same rate through the femoral vein (Fe-mice) and mice infused with a saline solution (Sal-mice) were used as controls. In Po-mice, hypoglycemia progressively developed until glucose levels dropped to a nadir of 2.3 +/- 0.1 mmol/l, whereas in Fe-mice, glycemia rapidly and transiently developed, and glucose levels increased to 7.7 +/- 0.6 mmol/l before progressively returning to fasting glycemic levels. Plasma insulin levels were similar in both Po- and Fe-mice during and at the end of the infusion periods (21.2 +/- 2.2 vs. 25.7 +/- 0.9 microU/ml, respectively, at 180 min of infusion). The whole-body glucose turnover rate was significantly higher in Po-mice than in Fe-mice (45.9 +/- 3.8 vs. 37.7 +/- 2.0 mg x kg(-1) x min)-1), respectively) and in Sal-mice (24.4 +/- 1.8 mg x kg(-1) x min(-1)). Somatostatin co-infusion with glucose in Po-mice prevented hypoglycemia without modifying the plasma insulin profile. Finally, tissue glucose clearance, which was determined after injecting 14C-2-deoxyglucose, increased to a higher level in Po-mice versus Fe-mice in the heart, brown adipose tissue, and the soleus muscle. Our data show that stimulation of the hepatoportal glucose sensor induced hypoglycemia and increased glucose utilization by a combination of insulin-dependent and insulin-independent or -sensitizing mechanisms. Furthermore, activation of the glucose sensor and/or transmission of its signal to target tissues can be blocked by somatostatin.

An adeno-associated virus-based intracellular sensor of pathological nuclear factor-κB activation for disease-inducible gene transfer.

Stimulation of resident cells by NF-κB activating cytokines is a central element of inflammatory and degenerative disorders of the central nervous system (CNS). This disease-mediated NF-κB activation could be used to drive transgene expression selectively in affected cells, using adeno-associated virus (AAV)-mediated gene transfer. We have constructed a series of AAV vectors expressing GFP under the control of different promoters including NF-κB -responsive elements. As an initial screen, the vectors were tested in vitro in HEK-293T cells treated with TNF-α. The best profile of GFP induction was obtained with a promoter containing two blocks of four NF-κB -responsive sequences from the human JCV neurotropic polyoma virus promoter, fused to a new tight minimal CMV promoter, optimally distant from each other. A therapeutical gene, glial cell line-derived neurotrophic factor (GDNF) cDNA under the control of serotype 1-encapsidated NF-κB -responsive AAV vector (AAV-NF) was protective in senescent cultures of mouse cortical neurons. AAV-NF was then evaluated in vivo in the kainic acid (KA)-induced status epilepticus rat model for temporal lobe epilepsy, a major neurological disorder with a central pathophysiological role for NF-κB activation. We demonstrate that AAV-NF, injected in the hippocampus, responded to disease induction by mediating GFP expression, preferentially in CA1 and CA3 neurons and astrocytes, specifically in regions where inflammatory markers were also induced. Altogether, these data demonstrate the feasibility to use disease-activated transcription factor-responsive elements in order to drive transgene expression specifically in affected cells in inflammatory CNS disorders using AAV-mediated gene transfer.