Common Gene Therapy Viral Vectors Do Not Efficiently Penetrate Sputum from Cystic Fibrosis Patients

Norwalk virus and human papilloma virus, two viruses that infect humans at mucosal surfaces, have been found capable of rapidly penetrating human mucus secretions. Viral vectors for gene therapy of Cystic Fibrosis (CF) must similarly penetrate purulent lung airway mucus (sputum) to deliver DNA to airway epithelial cells. However, surprisingly little is known about the rates at which gene delivery vehicles penetrate sputum, including viral vectors used in clinical trials for CF gene therapy. We find that sputum spontaneously expectorated by CF patients efficiently traps two viral vectors commonly used in CF gene therapy trials, adenovirus (d∼80 nm) and adeno-associated virus (AAV serotype 5; d∼20 nm), leading to average effective diffusivities that are ∼3,000-fold and 12,000-fold slower than their theoretical speeds in water, respectively. Both viral vectors are slowed by adhesion, as engineered muco-inert nanoparticles with diameters as large as 200 nm penetrate the same sputum samples at rates only ∼40-fold reduced compared to in pure water. A limited fraction of AAV exhibit sufficiently fast mobility to penetrate physiologically thick sputum layers, likely because of the lower viscous drag and smaller surface area for adhesion to sputum constituents. Nevertheless, poor penetration of CF sputum is likely a major contributor to the ineffectiveness of viral vector based gene therapy in the lungs of CF patients observed to date

Immune Response to Lactobacillus plantarum Expressing Borrelia burgdorferi OspA Is Modulated by the Lipid Modification of the Antigen

Over the past decade there has been increasing interest in the use of lactic acid bacteria as mucosal delivery vehicles for vaccine antigens, microbicides and therapeutics. We investigated the mechanism by which a mucosal vaccine based in recombinant lactic acid bacteria breaks the immunological tolerance of the gut in order to elicit a protective immune response.We analyzed how the lipid modification of OspA affects the localization of the antigen in our delivery vehicle using a number of biochemistry techniques. Furthermore, we examined how OspA-expressing L. plantarum breaks the oral tolerance of the gut by stimulating human intestinal epithelial cells, peripheral blood mononuclear cells and monocyte derived dendritic cells and measuring cytokine production. We show that the leader peptide of OspA targets the protein to the cell envelope of L. plantarum, and it is responsible for protein export across the membrane. Mutation of the lipidation site in OspA redirects protein localization within the cell envelope. Further, we show that lipidated-OspA-expressing L. plantarum does not induce secretion of the pro-inflammatory cytokine IL-8 by intestinal epithelial cells. In addition, it breaks oral tolerance of the gut via Th1/Th2 cell mediated immunity, as shown by the production of pro- and anti-inflammatory cytokines by human dendritic cells, and by the production of IgG2a and IgG1 antibodies, respectively.Lipid modification of OspA expressed in L. plantarum modulates the immune response to this antigen through a Th1/Th2 immune response

Gene therapy for carcinoma of the breast: Pro-apoptotic gene therapy

The dysregulation of apoptosis contributes in a variety of ways to the malignant phenotype. It is increasingly recognized that the alteration of pro-apoptotic and anti-apoptotic molecules determines not only escape from mechanisms that control cell cycle and DNA damage, but also endows the cancer cells with the capacity to survive in the presence of a metabolically adverse milieu, to resist the attack of the immune system, to locally invade and survive despite a lack of tissue anchorage, and to evade the otherwise lethal insults induced by drugs and radiotherapy. A multitude of apoptosis mediators has been identified in the past decade, and the roles of several of them in breast cancer have been delineated by studying the clinical correlates of pathologically documented abnormalities. Using this information, attempts are being made to correct the fundamental anomalies at the genetic level. Fundamental to this end are the design of more efficient and selective gene transfer systems, and the employment of complex interventions that are tailored to breast cancer and that are aimed concomitantly towards different components of the redundant regulatory pathways. The combination of such genetic modifications is most likely to be effective when combined with conventional treatments, thus robustly activating several pro-apoptotic pathways

Injection of a cholinergic agonist in the dorsolateral pontine tegmentum of cats affects the posturokinetic responses to cortical stimulation

International audienceMicroinjection into the dorsolateral pontine tegmentum of the muscarinic agonist bethanechol, leading to activation of cholinoceptive pontine reticular formation (pRF) neurons and the related medullary inhi-bitory reticulospinal system, did not modify the threshold, latency and amplitude of the forelimb flexion elicited by unilateral stimulation of the corresponding motor cortex. However, the remaining limbs which displayed a diagonal pattern of postural adjustments showed a dissociation of their postural responses in 2 components: the early component of central origin greatly decreased in amplitude, while the late component attributed to reflex mechanism triggered by the unbalance brought about by the flexion movement increased. Further evidence indicated that the pRF system intervenes in the gain regulation of the early postural responses during the cortically induced limb movement. Stimulation of the motor cortex produces in the cat not only a limb flexion, but also a diagonal pattern of postural adjustments characterized by a decreased force exerted by the limb diagonally opposite to the moving one and an increased force by the other two [6, 7]. The demonstration that the postural adjustments parallel the onset of movement ([8], see also [2, 6]) indicates that they are not of reflex origin, but result from a central command. The lateral vestibular nucleus (LVN), which exerts a direct excitatory influence on ipsilateral limb extensor motoneurons [14], represents a good candidate for such a role [9]. In fact, vestibulospinal (VS) neurons respond to stimulation of the contrala-teral motor cortex [13], an effect which disappears after partial cerebellectomy [11]. Moreover, lesion experiments indicate that the cortical influences responsible for the posturai adjustments during the limb movement utilize the precerebellar lateral reti-cular nucleus and the inferior olive [9, 17]

Cholinoceptive pontine reticular structures modify the postural adjustments during the limb movements induced by cortical stimulation.

International audience1. Activation of the pontine reticular formation (pRF) and the related medullary inhibitory reticulospinal (RS) system decreases the postural activity. This effect can be achieved either by local injection into the dorsal pontine tegmentum of cholinergic agonists which excite cholinoceptive pRF neurons, or by injection of noradrenergic agents which block the inhibitory influence exerted by the locus coeruleus (LC) neurons on the pRF. The main aim on the present study was to analyze the effects of tonic activation of these pRF neurons on the postural adjustments accompanying limb movements induced by motor cortex stimulation. In particular, electrodes were implanted chronically in the motor cortex of cats and stainless steel guide tubes of small size, later used for drug injection, were set bilaterally into sites just above the responsive regions. 2. Limb flexion elicited by stimulation of the motor cortex was accompanied by a diagonal pattern of postural adjustment, characterized by a decreased force exerted by the limb diagonally opposite to the moving one and an increased force exerted by the other two. 3. Microinjection into the pRF of both sides of 0.25 microliter of the muscarinic agonist bethanechol at the concentration of 8 or 16 micrograms/microliters in buffered artificial cerebrospinal fluid produced a short-lasting episode of postural atonia followed by a period of reduced postural activity, during which the cats were still able to stand on the measurement platform. Under this condition no changes in threshold, latency and amplitude of the flexion response were observed in the performing limb; however, the postural responses were considerably affected. In particular, when the performing limb was a forelimb, the other anterior limb showed a dissociation of the postural response in two distinct components. The first anticipatory component, which had a short latency (12-15 msec) and was considered to be centrally triggered, decreased in amplitude after injection of bethanechol and sometimes disappeared; on the other hand the second component, which had a long latency (50-60 msec) and was thus considered to be of reflex origin, increased in amplitude, due to the instability resulting from the depression of the early postural response. Similar results also affected to a lesser extent the hindlimbs. Moreover, body oscillations were observed and monitored from the force platforms following the late component of the postural responses.(ABSTRACT TRUNCATED AT 400 WORDS

Effects of changing stance conditions on anticipatory postural adjustment and reaction time to voluntary arm movement in humans

The effect on reaction time (RT) and anticipatory postural adjustment (APA) of unexpectedly changing stance conditions was studied using a push or pull arm movement task. The aim was to evaluate the modifiability of RT and APA by an external perturbation associated with an automatic compensatory reaction.Subjects standing on a moveable platform were asked to push or pull a rigid handle as quickly and as strongly as possible in response to the ‘go-signal’, a visual signal from a green or red light-emitting diode. Forward and backward translations of the platform were randomly induced at four time intervals after the go-signal. In some experiments to detect unspecific arousal there were no platform translations but an acoustic signal was given before the go-signal. Surface electromyographic activity (EMG) of upper arm and lower leg muscles was analysed.During the push task both RT and the duration of APA (onset of APA till the force signal indicating hand action) were shorter during backward than during forward translation. During the pull task the effect of platform translations was the reverse. The delay between go-signal and onset of APA remained constant. Consequently, RT and APA became shorter when the platform was translated in the same direction as that in which the upper body was displaced by the push or pull movement, and longer when it was translated in the opposite direction. The effects were maximal when translations were induced 250 ms after the go-signal, but a difference was detected up to 375 ms.Furthermore, with forward and backward platform translations RT was shorter when the translations were induced early rather than late after the go-signal. This was associated with a shortening of the delay between the go-signal and onset of APA, while APA duration remained constant. The shortened RT was in the range of that obtained when an acoustic signal was given just before the go-signal.It is concluded that (i) both the RT and the duration of APA can be modified by a translation of the support surface in a functionally appropriate way by updating the internal representation of the actual stance condition within the central nervous system. Both RT and APA are shortened when the body displacement induced by the push or pull movement and platform translation have the same direction; conversely, an inappropriate translation of the feet requires a greater APA and leads to a longer RT; (ii) both APA and RT are modifiable by platform translation for more than half the time between the go-signal and the focal push or pull movement; (iii) an unspecific effect of platform translation on RT can be identified; it may be mediated by a different neuronal substrate