Access to bacteriophage therapy: discouraging experiences from the human cell and tissue legal framework.

Cultures of human epithelial cells (keratinocytes) are used as an additional surgical tool to treat critically burnt patients. Initially, the production environment of keratinocyte grafts was regulated exclusively by national regulations. In 2004, the European Tissues and Cells Directive 2004/23/EC (transposed into Belgian Law) imposed requirements that resulted in increased production costs and no significant increase in quality and/or safety. In 2007, Europe published Regulation (EC) No. 1394/2007 on Advanced Therapy Medicinal Products. Overnight, cultured keratinocytes became (arguably) 'Advanced' Therapy Medicinal Products to be produced as human medicinal products. The practical impact of these amendments was (and still is) considerable. A similar development appears imminent in bacteriophage therapy. Bacteriophages are bacterial viruses that can be used for tackling the problem of bacterial resistance development to antibiotics. Therapeutic natural bacteriophages have been in clinical use for almost 100 years. Regulators today are framing the (re-)introduction of (natural) bacteriophage therapy into 'modern western' medicine as biological medicinal products, also subject to stringent regulatory medicinal products requirements. In this paper, we look back on a century of bacteriophage therapy to make the case that therapeutic natural bacteriophages should not be classified under the medicinal product regulatory frames as they exist today. It is our call to authorities to not repeat the mistake of the past.

A prospective multicenter study of the efficacy and tolerability of cryopreserved allogenic human keratinocytes to treat venous leg ulcers.

Allogeneic human keratinocyte cultures have been used to treat burn wounds, donor sites, and chronic skin ulcers with some success. Cryopreservation of these cultures allows for the production of large standardized batches that are readily available for use. The aim of the study presented in this report was to study effects of cryopreserved cultured allogenic human keratinocytes (CryoCeal) on chronic lower extremity wounds. Parameters were measured to study efficacy, tolerability, pain associated with chronic wounds, and quality of life of patients. Twenty-seven patients with hard-to-heal venous leg ulcers received a maximum of 9 applications of CryoCeal in a prospective, uncontrolled multicenter study lasting 48 weeks. Eleven out of 27 patients (41%; 95% CI: 22%-61%) had complete wound closure within 24 weeks (1 week). The time required for complete wound closure in these 11 patients ranged from 4.1 to 24.9 weeks. Only 1 patient had recurrence of the ulcer at 48 weeks. Local (wound) pain scores decreased from a mean of 2.5 at baseline to 0.9 at week 24. Fifty percent of the patients attained a pain score of 0 after 12 weeks and remained stable at this score until the end of the study. Overall, the patient quality of life was better at week 24, compared to baseline values. The treatment was well tolerated, and wound infection was the most frequently occurring adverse event.

A dynamic recurrent neural network for multiple muscles electromyographic mapping to elevation angles of the lower limb in human locomotion.

This paper describes the use of a dynamic recurrent neural network (DRNN) for simulating lower limb coordination in human locomotion. The method is based on mapping between the electromyographic signals (EMG) from six muscles and the elevation angles of the three main lower limb segments (thigh, shank and foot). The DRNN is a fully connected network of 35 hidden units taking into account the temporal relationships history between EMG and lower limb kinematics. Each EMG signal is sent to all 35 units, which converge to three outputs. Each output neurone provides the kinematics of one lower limb segment. The training is supervised, involving learning rule adaptations of synaptic weights and time constant of each unit. Kinematics of the locomotor movements were recorded and analysed using the opto-electronic ELITE system. Comparative analysis of the learning performance with different types of output (position, velocity and acceleration) showed that for common gait mapping velocity data should be used as output, as it is the best compromise between asymptotic error curve, rapid convergence and avoidance of bifurcation. Reproducibility of the identification process and biological plausibility were high, indicating that the DRNN may be used for understanding functional relationships between multiple EMG and locomotion. The DRNN might also be of benefit for prosthetic control.

Development of a kinematic coordination pattern in toddler locomotion: planar covariation.

The purpose of this study is to analyze the coordination patterns of the elevation angles of lower limb segments following the onset of unsupported walking in children and to look for the existence of a planar covariation rule as previously described in adult human locomotion. The kinematic patterns of locomotion were recorded in 21 children (11-144 months of age) and 19 adults. In 4 children we monitored the very first unsupported steps. The extent to which the covariation of thigh, shank, and foot angles was constrained on a plane in 3D space was assessed by means of orthogonal regression and statistically quantified by means of principal component analysis. The orientation of the covariation plane of the children was compared with the mean value of the adults' plane. Trunk stability with respect to the vertical was assessed in both the frontal (roll) and sagittal (pitch) planes. The evolution with walking experience of the plane orientation and trunk oscillations demonstrated biexponential profiles with a relatively fast time constant (< 6 months after the onset of unsupported locomotion) followed by a much slower progression toward adult values. The initial fast changes of these walking parameters did not parallel the slow, monotonic maturation of anthropometric parameters. The early emergence of the covariation plane orientation and its correlation with trunk vertical stability reflect the dynamic integration of postural equilibrium and forward propulsion in a gravity-centered frame. The results support the view that the planar covariation reflects a coordinated, centrally controlled behavior, in addition to biomechanical constraints. The refinement of the planar covariation while morphological variables drastically change as the child grows implies a continuous update of the neural command.

Kinematics invariance in multi-directional complex movements in free space: effect of changing initial direction.

We investigated in normal human subjects the effect of changing the initial direction on the kinematic properties of figure '8' movement performed as fast as possible by the right arm extended in free space. To this end, the motion of the index finger was monitored by the ELITE system. The figure '8' movement was characterized by a complex tangential velocity profile (Vt) presenting 5 bell-shaped components. It was found that the temporal segmentation following Vt was not significantly different, whatever the initial direction of the movement. The decomposition of Vt into different velocity profiles with respect to vertical (3 phases, Iy-IIIy) and horizontal (5 phases, Iz-Vz) directions showed a significant relationship between the amplitude and the maximal velocity for all the different phases (except the IIy phase), which demonstrated a good conservation of the Isochrony Principle. However, we showed that the transition between the clockwise and counter-clockwise loop (inflection point) induced greater variability in the vertical velocity profile than in the horizontal one. Moreover, some parameters such as the maximal velocity of Iy and the movement amplitude of the last phases (IIIy and Vz) showed significant changes depending on the initial direction. A highly significant positive correlation was observed between the instantaneous curvature and angular velocity. This was expressed by a power law similar to that previously describe for other types of movement. Furthermore, it was found that this covariation between geometrical and kinematic properties of the trajectory is not dependent on the initial direction of movement. In conclusion, these results support the idea that the fast execution in different directions of a figure '8' movement is mainly controlled by two types of invariant commands. The first one is reflected in the 2/3 power law between angular velocity and curvature and the second one is represented by a segmented tangential velocity profile.

In vitro and in vivo biocompatibility of dextran dialdehyde cross-linked gelatin hydrogel films.

The biosafety of a new hydrogel wound dressing material consisting of dextran dialdehyde cross-linked gelatin was evaluated (i) in vitro in cultures of dermal fibroblasts, epidermal keratinocytes, and endothelial cells, three cell types which play a major role in the process of cutaneous wound healing, and (ii) in vivo by subcutaneous implantation studies in mice. The cytotoxicities of this hydrogel, two semi-occlusive polyurethane dressings (Tegaderm and OpSite), and a hydrocolloid dressing (DuoDERM) were compared by measuring cell survival with the tetrazolium salt reduction (MTT) assay after incubations of the wound dressing samples for up to 6 d, in the presence of--but not in direct contact with--the cells. In vitro, the degree of cytotoxicity of the new hydrogel was greater in keratinocyte cultures than in fibroblast and endothelial cell cultures, and increased upon longer incubation time. In keratinocyte cultures, the semi-occlusive polyurethane dressings, the hydrocolloid, and the hydrogel dressings induced low, high and acceptable degrees of cytotoxicity, respectively. The toxicity of the isolated hydrogel components was assessed in Balb MK keratinocyte cultures. In these cells, epidermal growth-factor-stimulated thymidine incorporation into DNA was higher in the presence of gelatin. By contrast, concentrations of dextran dialdehyde as low as 0.002% were found to significantly decrease thymidine incorporation (P < 0.01). Subcutaneous implantation studies in mice showed that in vivo the hydrogel was biocompatible since the foreign body reaction seen around the implanted hydrogel samples was moderate and became minimal upon increasing implantation time. These results indicate that dextran dialdehyde cross-linked gelatin hydrogels have an appropriate biocompatibility.

In vitro release characteristics of bioactive molecules from dextran dialdehyde cross-linked gelatin hydrogel films.

Hydrogel films, prepared by cross-linking of gelatin with dextran dialdehydes (weight ratio 2:1), and containing either fluorescein isothiocyanate dextran (Mw 70000) or polypeptides were evaluated in terms of their release characteristics and mechanical properties upon increasing storage time at 4 degrees C. Important changes in release kinetics and mechanical properties of the cross-linked gelatin films were observed, especially during the first week after the hydrogel production. Rheological and NMR measurements showed that the mechanical properties of the gelatin hydrogel films were improved with increasing storage time. It appeared that the process of chemical cross-linking and physical structuring of the gelatin hydrogel matrix did not occur instantaneously and substantially influenced the polypeptide release patterns. Cross-linked gelatin hydrogels were found to be appropriate release systems for medium-term sustained delivery of biologically active epidermal growth factor (EGF), but release characteristics were strongly dependent on the nature of the protein which was incorporated.

Multi-joint coordination strategies for straightening up movement in humans.

Complex movement execution theoretically involves numerous biomechanical degrees of freedom, leading to the concept of redundancy. The kinematics and kinetics of rapid straightening up movement from the squatting position were analysed with the optoelectronic ELITE system in 14 subjects. We found multiple acceleration and deceleration peaks for the hip, knee and ankle joints during the early extension phase of the movement. In order to test the temporal coordination between the angular acceleration of these joints, conjugate crosscorrelation functions (CCF) between each set of two variables were calculated. We found a bimodal distribution of the maximum CCF in positive and negative values suggesting the existence of two distinct strategies, the in-phase and the out-of-phase strategy for each pair of joints. The hip and knee coordination strategies (in- or out-of-phase) were well conserved in each subject for repetitive movements. Combination of joint pair strategies was more reproducible for the hip-knee/knee-ankle pair than for the other combinations, suggesting that the straightening up strategies are organised around the knee. We conclude that mastering of the redundancy problem can be realised by using coordination strategies characterised by opposed joint acceleration patterns.

Emergence of clusters in the hidden layer of a dynamic recurrent neural network.

The neural integrator of the oculomotor system is a privileged field for artificial neural network simulation. In this paper, we were interested in an improvement of the biologically plausible features of the Arnold-Robinson network. This improvement was done by fixing the sign of the connection weights in the network (in order to respect the biological Dale's Law). We also introduced a notion of distance in the network in the form of transmission delays between its units. These modifications necessitated the introduction of a general supervisor in order to train the network to act as a leaky integrator. When examining the lateral connection weights of the hidden layer, the distribution of the weights values was found to exhibit a conspicuous structure: the high-value weights were grouped in what we call clusters. Other zones are quite flat and characterized by low-value weights. Clusters are defined as particular groups of adjoining neurons which have strong and privileged connections with another neighborhood of neurons. The clusters of the trained network are reminiscent of the small clusters or patches that have been found experimentally in the nucleus prepositus hypoglossi, where the neural integrator is located. A study was conducted to determine the conditions of emergence of these clusters in our network: they include the fixation of the weight sign, the introduction of a distance, and a convergence of the information from the hidden layer to the motoneurons. We conclude that this spontaneous emergence of clusters in artificial neural networks; performing a temporal integration, is due to computational constraints, with a restricted space of solutions. Thus, information processing could induce the emergence of iterated patterns in biological neural networks.

Evidence of a preprogrammed deactivation of the hamstring muscles for triggering rapid changes of posture in humans.

Normal subjects were asked to make rapid flexions of the legs from a stationary initial standing posture in a self-paced mode. Because this movement implicates a rapid change in posture, questions were asked about the type of central command which must include the rupture of the erect posture and the accomplishment of the goal directed movement. Movements of the different segments of the body were recorded and analyzed using the optoelectronic ELITE system. Electromyographic (EMG) activities of 8 muscles of the lower limb on one side were recorded, rectified and integrated. The time relationships of the different EMG signals (activation or deactivation) were analyzed with respect to selected kinetic measures of the related segments of the body. In the majority of the subjects, before the movement onset, EMG events included a specific deactivation of the tonic EMG activity of the semimembranous (SM) and semitendinous (ST) muscles (time onset relative to the onset of the legs flexion: -196.9 +/- 96.4 ms and -180.5 +/- 89.7 ms, respectively). A second event was a phasic activation of the tibialis anterior (TA) muscle (time onset: -60.5 +/- 117.6 ms). Conjugate cross-correlation analysis of these EMG signals demonstrated the existence of a common coordinated strategy between the deactivation of the hamstring and the TA activation. Even though a small horizontal displacement of the head was recorded prior to leg movement, it occurred too late to induce deactivation of the hamstring muscles. These results demonstrate that for rapid legs flexion, where the gravity forces are the main source of joint angle acceleration, the deactivation of the SM and ST muscles acts in conjunction with the phasic activation of the TA. The preprogrammed deactivation of the SM and ST muscles represents the early phase of the central command to switch from the standing to the squatting posture.

Healing of full-thickness wounds in pigs: effects of occlusive and non-occlusive dressings associated with a gel vehicle.

This study, based upon a pig model, was conducted to investigate the effects of moist and dry healing conditions on wound closure (epithelialization, granulation tissue, contraction) of full-thickness wounds. Thirty-two full-thickness square wounds (3 cm x 3 cm) covered with either an occlusive polyurethane dressing (Tegaderm) or a non-occlusive dressing (Melolin) were evaluated. The effect of the presence or the absence of a gel (3% Idroramnosan) was also investigated with both dressings. The dressings were renewed twice a week. The time required for wound closure was 19.2 +/- 1.6 days for Tegaderm and 26.6 +/- 3.0 days (means +/- SD) for Melolin, respectively. The healing time of the full-thickness porcine wounds was significantly (P < 0.001) reduced by the occlusive dressing. Equivalent results were found with the 3% gel, indicating that the gel can be used as a neutral vehicle. The healing rate, calculated according to Gilman's method, was also significantly (P < 0.001) enhanced by the occlusive dressing. This progression was 0.073 +/- 0.004 cm/day and 0.050 +/- 0.009 cm/day (means +/- SD) for Tegaderm and Melolin, respectively. The contribution of contraction to wound closure was similar in all wounds, indicating that the occlusive dressing did not have an effect on wound contraction. Histological evaluation was performed on full-thickness skin biopsies of whole wound harvested from the time of wound closure to 3 months after. At any time point, no significant histological variations were observed between the different treated wounds. This study demonstrates in a porcine model that for full-thickness wounds, as for split-thickness wounds, occlusive dressing enhances healing rate and shortens the time for wound repair. The shortened healing time is a function primarily of the effect of occlusive dressing on epithelialization, especially the third phase of wound resurfacing.

A dynamic neural network identification of electromyography and arm trajectory relationship during complex movements.

We propose a new approach based on dynamic recurrent neural networks (DRNN) to identify, in human, the relationship between the muscle electromyographic (EMG) activity and the arm kinematics during the drawing of the figure eight using an extended arm. After learning, the DRNN simulations showed the efficiency of the model. We demonstrated its generalization ability to draw unlearned movements. We developed a test of its physiological plausibility by computing the error velocity vectors when small artificial lesions in the EMG signals were created. These lesion experiments demonstrated that the DRNN has identified the preferential direction of the physiological action of the studied muscles. The network also identified neural constraints such as the covariation between geometrical and kinematics parameters of the movement. This suggests that the information of raw EMG signals is largely representative of the kinematics stored in the central motor pattern. Moreover, the DRNN approach will allow one to dissociate the feedforward command (central motor pattern) and the feedback effects from muscles, skin and joints.

Healing of full-thickness wounds treated with lyophilized cultured keratinocyte cell lysate in genetically diabetic mice.

The effect of a lyophilized cell lysate prepared from cultured human keratinocytes on the healing of full-thickness wounds was evaluated in an impaired healing model. Full-thickness wounds (8 mm in diameter) were made on the dorsal areas of female genetically diabetic mice C57 BL/KsJ (db/db) and their normal (db/+) littermates. Wounds were covered with an occlusive polyurethane film dressing and were treated for 5 days either with the lyophilized cell lysate from cultured human keratinocytes prepared in phosphate-buffered saline solution or with phosphate-buffered saline solution. In normal (db/+) mice, all wounds were closed 16 days after wounding, and more than 90% of the wound closure was due to wound contraction. Wound contraction accounted for a similar extent of wound closure in both lyophilized cell lysate-treated and phosphate-buffered saline solution-treated wounds. In contrast, in the diabetic (db/db) mice, after histologic examination of the wounds 32 days after wounding, four of ten lyophilized cell lysate-treated wounds and four of seven phosphate-buffered saline-treated wounds were found to be closed. Moreover, applications of lyophilized cell lysate from cultured human keratinocytes to full-thickness wounds in diabetic db/db mice significantly decreased the contribution of contraction to wound closure. Day 32 after wounding, contraction contribution to wound closure amounted to 57.7%+/- 4.7% and 80.4%+/- 3.2% (mean +/- standard error of the mean, p < 0.005) of the initial wound areas, respectively, for lyophilized cell lysate-treated and phosphate-buffered saline solution-treated wounds. At this time of wound healing, the thickness of the dermis was increased 1.7-fold by the keratinocyte cell lysate treatment, but neither epithelial migration from the wound edges nor the thickness of the regenerated epithelium were significantly affected. In conclusion, in diabetic (db/db) mice the application of lyophilized cell lysate from cultured human keratinocytes influenced the healing of the dermis and wound contraction, but had no effect on reepithelialization.

Bile acids and conjugates identified in metabolic disorders by fast atom bombardment and tandem mass spectrometry.

From a study of the collision-activated fragmentation of bile acids, a qualitative analytical method based on negative ion fast atom bombardment tandem mass spectrometry has been developed. The times for sample preparation and analyses are short. Both free and conjugated bile acids are detected as they occur in biological fluids, without derivatization. For identifying bile acids and conjugates, the method offers better specificity and sensitivity than does the fast atom bombardment mass spectrometric technique alone. Specific scan modes have been developed for the selective detection of taurine conjugates, delta 4-unsaturated taurine conjugates, delta 4-3-keto free acids and their glycine conjugates, free acids and glycine conjugates bearing a hydroxyl group at the C-12 position, sulfates of glycine and taurine conjugates, and a C29 dicarboxylic bile acid, specific for generalized peroxisomal disorders. Applications of this technique demonstrate its potential usefulness, principally in the diagnosis of several peroxisomal disorders.

Influence of chronic administration of valproate on ultrastructure and enzyme content of peroxisomes in rat liver and kidney. Oxidation of valproate by liver peroxisomes.

Chronic administration to rats of the anticonvulsant drug, valproate, induced proliferation of liver peroxisomes and selectively increased the activity of the enzymes involved in beta-oxidation in these organelles. In kidney cortex, only a moderate increase in enzyme activity could be recorded. Valproate (1% w/w in the diet for 25 to 100 days) caused the appearance on electron micrographs of unusual tubular inclusions in the matrix of liver peroxisomes. SDS-PAGE analysis of purified peroxisomal fractions from treated rats demonstrated an increase in the content of five polypeptides; four of which most likely correspond to enzymes of the peroxisomal beta-oxidation. It is suggested that the peroxisomal inclusions correspond to the accumulation of these polypeptides in the matrix of the organelle. An in vivo evaluation of the peroxisomal hydrogen peroxide production suggested that valproate itself or one of its metabolites is substrate for peroxisomal beta-oxidation. This was confirmed by in vitro studies. Activation of valproate or its metabolites by liver acyl-CoA synthetase could be demonstrated, although it was 50 times slower than that of octanoate. This reaction further led to a small, but significant production of H2O2 by the action of peroxisomal acyl-CoA oxidase.

Study of reactions induced by hydroxylamine treatment of esters of organic acids and of 3-ketoacids: application to the study of urines from patients under valproate therapy.

Hydroxylamine used at alkaline pH as oximating agent in the search for organic aciduria by gas chromatography/mass spectrometry (GC/MS) induces other chemical reactions. Esters are partially transformed in their corresponding hydroxamic acids. GC/MS characteristics of the trimethylsilylated derivatives of the hydroxamic acids arising from alpha-unsaturated esters are here reported. Their mass spectral fragmentation helps in the recognition of peaks arising from the glucuronides of 2-ene- and probably 2,3'-diene-valproic acid. By heating in the injection port of the gas chromatograph, part of some trimethylsilylated hydroxamic acids are transformed to the corresponding isocyanates by a Lossen-like rearrangement. In addition to the corresponding hydroxamic acids, hydroxylamine treatment of alpha-unsaturated esters forms 2-isoxazolidin-3-ones by intramolecular Michael addition. GC/MS characteristics of the trimethylsilylated derivatives of these compounds are reported. Submitted to hydroxylamine, 3-ketoacids forms 2-isoxazolin-5-ones by cyclization of the oximes after acidification. This explains the existence of two GC peaks observed from urine extracts of patients under valproate therapy, which correspond to two tautomers of 2-isoxazolin-5-one originating from the oximes of the 3-keto-valproic acid.