Harmonic Imaging of Stem Cells in Whole Blood at GHz Pixel Rate.

The pre-clinical validation of cell therapies requires monitoring the biodistribution of transplanted cells in tissues of host organisms. Real-time detection of these cells in the circulatory system and identification of their aggregation state is a crucial piece of information, but necessitates deep penetration and fast imaging with high selectivity, subcellular resolution, and high throughput. In this study, multiphoton-based in-flow detection of human stem cells in whole, unfiltered blood is demonstrated in a microfluidic channel. The approach relies on a multiphoton microscope with diffractive scanning in the direction perpendicular to the flow via a rapidly wavelength-swept laser. Stem cells are labeled with metal oxide harmonic nanoparticles. Thanks to their strong and quasi-instantaneous second harmonic generation (SHG), an imaging rate in excess of 10 000 frames per second is achieved with pixel dwell times of 1 ns, a duration shorter than typical fluorescence lifetimes yet compatible with SHG. Through automated cell identification and segmentation, morphological features of each individual detected event are extracted and cell aggregates are distinguished from isolated cells. This combination of high-speed multiphoton microscopy and high-sensitivity SHG nanoparticle labeling in turbid media promises the detection of rare cells in the bloodstream for assessing novel cell-based therapies.

Biofilm formation of the food spoiler Brochothrix thermosphacta on different industrial surface materials using a biofilm reactor.

Brochothrix thermosphacta is considered as a major food spoiler bacteria. This study evaluates biofilm formation by B. thermosphacta CD337(2) - a strong biofilm producer strain - on three food industry materials (polycarbonate (PC), polystyrene (PS), and stainless steel (SS)). Biofilms were continuously grown under flow at 25 °C in BHI broth in a modified CDC biofilm reactor. Bacterial cells were enumerated by plate counting, and biofilm spatial organization was deciphered by combining confocal laser scanning microscopy and image analysis. The biofilms had the same growth kinetics on all three materials and reach 8log CFU/cm2 as maximal concentration. Highly structured biofilms were observed on PC and PS, but less structured ones on SS. This difference was confirmed by structural quantification analysis using the image analysis software tool BiofilmQ. Biofilm on SS show less roughness, density, thickness and volume. The biofilm 3D structure seemed to be related to the coupon topography and roughness. The materials used in this study do not affect biofilm growth. However, their roughness and topography affect the biofilm architecture, which could influence biofilm behaviour.

Label-free 3D characterization of cardiac fibrosis in muscular dystrophy using SHG imaging of cleared tissue.

BACKGROUND INFORMATION: Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by mutations in the gene encoding dystrophin. It leads to repeated cycles of muscle fiber necrosis and regeneration and progressive replacement of fibers by fibrotic and adipose tissue, with consequent muscle weakness and premature death. Fibrosis and, in particular, collagen accumulation are important pathological features of dystrophic muscle. A better understanding of the development of fibrosis is crucial to enable better management of DMD. Three-dimensional (3D) characterization of collagen organization by second harmonic generation (SHG) microscopy has already proven a highly informative means of studying the fibrotic network in tissue. RESULTS: Here, we combine for the first-time tissue clearing with SHG microscopy to characterize in depth the 3D cardiac fibrosis network from DMDmdx rat model. Heart sections (1-mm-thick) from 1-year-old wild-type (WT) and DMDmdx rats were cleared using the CUBIC protocol. SHG microscopy revealed significantly greater collagen deposition in DMDmdx versus WT sections. Analyses revealed a specific pattern of SHG+ segmented objects in DMDmdx cardiac muscle, characterized by a less elongated shape and increased density. Compared with the observed alignment of SHG+ collagen fibers in WT rats, profound fiber disorganization was observed in DMDmdx rats, in which we observed two distinct SHG+ collagen fiber profiles, which may reflect two distinct stages of the fibrotic process in DMD. CONCLUSION AND SIGNIFICANCE: The current work highlights the interest to combine multiphoton SHG microscopy and tissue clearing for 3D fibrosis network characterization in label free organ. It could be a relevant tool to characterize the fibrotic tissue remodeling in relation to the disease progression and/or to evaluate the efficacy of therapeutic strategies in preclinical studies in DMD model or others fibrosis-related cardiomyopathies diseases.

Skeletal Muscle Regenerative Potential of Human MuStem Cells following Transplantation into Injured Mice Muscle.

After intra-arterial delivery in the dystrophic dog, allogeneic muscle-derived stem cells, termed MuStem cells, contribute to long-term stabilization of the clinical status and preservation of the muscle regenerative process. However, it remains unknown whether the human counterpart could be identified, considering recent demonstrations of divergent features between species for several somatic stem cells. Here, we report that MuStem cells reside in human skeletal muscle and display a long-term ability to proliferate, allowing generation of a clinically relevant amount of cells. Cultured human MuStem (hMuStem) cells do not express hematopoietic, endothelial, or myo-endothelial cell markers and reproducibly correspond to a population of early myogenic-committed progenitors with a perivascular/mesenchymal phenotypic signature, revealing a blood vessel wall origin. Importantly, they exhibit both myogenesis in vitro and skeletal muscle regeneration after intramuscular delivery into immunodeficient host mice. Together, our findings provide new insights supporting the notion that hMuStem cells could represent an interesting therapeutic candidate for dystrophic patients.

Label-Free Fried Starchy Matrix: Investigation by Harmonic Generation Microscopy.

An innovative methodology based on non-destructive observation by using harmonic generation microscopy is proposed for detection and location of starch granules and oil in a fried starchy matrix and topography analysis of food products. Specific fluorescent probes were used to label the main biochemical components of the starchy fried matrix, namely starch and oil. Fluorescence of starch and oil respectively stained with Safranin O and Nile red was observed from non-linear microscopy. By using sequential scanning and specific emission filters, it was possible to merge fluorescence and harmonic generation signals. Second harmonic generation (SHG) generated by starch granules was superposed with safranin fluorescence, whereas third harmonic generation (THG), not restricted to the superposition with Nile red fluorescent signal, was used to investigate the topography of the fried product. By these experiments, starch granule mapping and topography of the starchy fried product were obtained without any destructive preparation of the sample. This label-free approach using harmonic generation microscopy is a very promising methodology for microstructure investigation of a large panel of starchy food products.

Quantitative proteome profiling of dystrophic dog skeletal muscle reveals a stabilized muscular architecture and protection against oxidative stress after systemic delivery of MuStem cells.

Proteomic profiling plays a decisive role in the elucidation of molecular signatures representative of a specific clinical context. MuStem cell based therapy represents a promising approach for clinical applications to cure Duchenne muscular dystrophy (DMD). To expand our previous studies collected in the clinically relevant DMD animal model, we decided to investigate the skeletal muscle proteome 4 months after systemic delivery of allogenic MuStem cells. Quantitative proteomics with isotope-coded protein labeling was used to compile quantitative changes in the protein expression profiles of muscle in transplanted Golden Retriever muscular dystrophy (GRMD) dogs as compared to Golden Retriever muscular dystrophy dogs. A total of 492 proteins were quantified, including 25 that were overrepresented and 46 that were underrepresented after MuStem cell transplantation. Interestingly, this study demonstrates that somatic stem cell therapy impacts on the structural integrity of the muscle fascicle by acting on fibers and its connections with the extracellular matrix. We also show that cell infusion promotes protective mechanisms against oxidative stress and favors the initial phase of muscle repair. This study allows us to identify putative candidates for tissue markers that might be of great value in objectively exploring the clinical benefits resulting from our cell-based therapy for DMD. All MS data have been deposited in the ProteomeXchange with identifier PXD001768 (http://proteomecentral.proteomexchange.org/dataset/PXD001768).

Identification in GRMD dog muscle of critical miRNAs involved in pathophysiology and effects associated with MuStem cell transplantation.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked muscle disease that leads to fibre necrosis and progressive paralysis. At present, DMD remains a lethal disease without any effective treatment, requiring a better understanding of the pathophysiological processes and comprehensive assessment of the newly identified therapeutic strategies. MicroRNAs including members of the muscle-specific myomiR family have been identified as being deregulated in muscle of DMD patients and in mdx mice used as a model for DMD. In recent years, the Golden Retriever muscular dystrophy (GRMD) dog has appeared as the crucial animal model for objectively assessing the potential of new innovative approaches. Here, we first aim at establishing the muscle expression pattern of five selected miRNAs in this clinically relevant model to determine if they are similarly affected compared with other DMD contexts. Second, we attempt to show whether these miRNAs could be impacted by the systemic delivery of a promising stem cell candidate (referred to as MuStem cells) to implement our knowledge on its mode of action and/or identify markers associated with cell therapy efficacy. METHODS: A comparative study of miRNAs expression levels and cellular localization was performed on 9-month-old healthy dogs, as well as on three sub-sets of GRMD dog (without immunosuppression or cell transplantation, with continuous immunosuppressive regimen and with MuStem cell transplantation under immunosuppression), using RT-qPCR and in situ hybridization. RESULTS: We find that miR-222 expression is markedly up-regulated in GRMD dog muscle compared to healthy dog, while miR-486 tends to be down-expressed. Intriguingly, the expression of miR-1, miR-133a and miR-206 does not change. In situ hybridization exploration reveals, for the first time, that miR-486 and miR-206 are mainly localized in newly regenerated fibres in GRMD dog muscle. In addition, we show that cyclosporine-based immunosuppression, classically used in allogeneic cell transplantation, exclusively impacts the miR-206 expression. Finally, we demonstrate that intra-arterial administration of MuStem cells results in up-regulation of miR-133a and miR-222 concomitantly with a down-expression of two sarcomeric proteins corresponding to miR-222 targets. CONCLUSION: We point out a differential muscle expression of miR-222 and miR-486 associated with the pathophysiology of the clinically relevant GRMD dog model with a tissue localization focused on regenerated fibres. We also establish a modified expression of miR-133a and miR-222 subsequent to MuStem cell infusion.

ß2-Adrenoreceptor agonist inhibits antigen cross-presentation by dendritic cells.

Despite widespread usage of ß-adrenergic receptor (AR) agonists and antagonists in current clinical practice, our understanding of their interactions with the immune system is surprisingly sparse. Among the AR expressed by dendritic cells (DC), ß2-AR can modify in vitro cytokine release upon stimulation. Because DC play a pivotal role in CD8(+) T cell immune responses, we examined the effects of ß2-AR stimulation on MHC class I exogenous peptide presentation and cross-presentation capacities. We demonstrate that ß2-AR agonist-exposed mature DC display a reduced ability to cross-present protein Ags while retaining their exogenous peptide presentation capability. This effect is mediated through the nonclassical inhibitory G (Gαi/0) protein. Moreover, inhibition of cross-presentation is neither due to reduced costimulatory molecule expression nor Ag uptake, but rather to impaired phagosomal Ag degradation. We observed a crosstalk between the TLR4 and ß2-AR transduction pathways at the NF-κB level. In vivo, ß2-AR agonist treatment of mice inhibits Ag protein cross-presentation to CD8(+) T cells but preserves their exogenous MHC class I peptide presentation capability. These findings may explain some side effects on the immune system associated with stress or ß-agonist treatment and pave the way for the development of new immunomodulatory strategies.

Multiphoton microscopy is a nondestructive label-free approach to investigate the 3D structure of gas cell walls in bread dough.

During the different steps of bread-making, changes in the microstructure of the dough, particularly in the gas cell walls (GCW), have a major influence on the final bread crumb texture. Investigation of the spatial conformation of GCWs is still a challenge because it requires both high resolutions and 3D depth imaging. The originality of the present work lies in the use of label-free non-destructive multiphoton microscopy (NLOM) to image the 3D structure of GCWs, shedding light on their behavior and organization in wheat bread dough. We demonstrated that second and third harmonic generation (SHG, THG) allow imaging, respectively, of starch granules and interfaces in bread dough, while the gluten matrix was detected via two-photon excitation fluorescence (TPEF). Last, a distinction between the gluten network and starch granules was achieved using gluten endogenous fluorescence (EF) imaging, while the position, size, and 3D orientation of starch granules in GCWs were determined from harmonic imaging, made possible by the acquisition of backward and forward SHG with linear polarization. These innovative experiments highlight the strengths of NLOM for a label-free characterization of bread dough microstructure for the first time, in order to understand the role of starch granules in dough stabilization.

Specific and label-free endogenous signature of dystrophic muscle by Synchrotron deep ultraviolet radiation.

Dystrophic muscle is characterized by necrosis/regeneration cycles, inflammation, and fibro-adipogenic development. Conventional histological stainings provide essential topographical data of this remodeling but may be limited to discriminate closely related pathophysiological contexts. They fail to mention microarchitecture changes linked to the nature and spatial distribution of tissue compartment components. We investigated whether label-free tissue autofluorescence revealed by Synchrotron deep ultraviolet (DUV) radiation could serve as an additional tool for monitoring dystrophic muscle remodeling. Using widefield microscopy with specific emission fluorescence filters and microspectroscopy defined by high spectral resolution, we analyzed samples from healthy dogs and two groups of dystrophic dogs: naïve (severely affected) and MuStem cell-transplanted (clinically stabilized) animals. Multivariate statistical analysis and machine learning approaches demonstrated that autofluorescence emitted at 420-480 nm by the Biceps femoris muscle effectively discriminates between healthy, dystrophic, and transplanted dog samples. Microspectroscopy showed that dystrophic dog muscle displays higher and lower autofluorescence due to collagen cross-linking and NADH respectively than that of healthy and transplanted dogs, defining biomarkers to evaluate the impact of cell transplantation. Our findings demonstrate that DUV radiation is a sensitive, label-free method to assess the histopathological status of dystrophic muscle using small amounts of tissue, with potential applications in regenerative medicine.

Efficient intracerebral delivery of AAV5 vector encoding human ARSA in non-human primate.

Metachromatic leukodystrophy (MLD) is a lethal neurodegenerative disease caused by a deficiency in the lysosomal arylsulfatase A (ARSA) enzyme leading to the accumulation of sulfatides in glial and neuronal cells. We previously demonstrated in ARSA-deficient mice that intracerebral injection of a serotype 5 adeno-associated vector (AAV) encoding human ARSA corrects the biochemical, neuropathological and behavioral abnormalities. However, before considering a potential clinical application, scaling-up issues should be addressed in large animals. Therefore, we performed intracerebral injection of the same AAV vector (total dose of 3.8 x 10(11) or 1.9 x 10(12) vector genome, three sites of injection in the right hemisphere, two deposits per site of injection) into three selected areas of the centrum semiovale white matter, or in the deep gray matter nuclei (caudate nucleus, putamen, thalamus) of six non-human primates to evaluate vector distribution, as well as expression and activity of human ARSA. The procedure was perfectly tolerated, without any adverse effect or change in neurobehavioral examination. AAV vector was detected in a brain volume of 12-15 cm(3) that corresponded to 37-46% of the injected hemisphere. ARSA enzyme was expressed in multiple interconnected brain areas over a distance of 22-33 mm. ARSA activity was increased by 12-38% in a brain volume that corresponded to 50-65% of injected hemisphere. These data provide substantial evidence for potential benefits of brain gene therapy in patients with MLD.

Systemic delivery of allogenic muscle stem cells induces long-term muscle repair and clinical efficacy in duchenne muscular dystrophy dogs.

Duchenne muscular dystrophy (DMD) is a genetic progressive muscle disease resulting from the lack of dystrophin and without effective treatment. Adult stem cell populations have given new impetus to cell-based therapy of neuromuscular diseases. One of them, muscle-derived stem cells, isolated based on delayed adhesion properties, contributes to injured muscle repair. However, these data were collected in dystrophic mice that exhibit a relatively mild tissue phenotype and clinical features of DMD patients. Here, we characterized canine delayed adherent stem cells and investigated the efficacy of their systemic delivery in the clinically relevant DMD animal model to assess potential therapeutic application in humans. Delayed adherent stem cells, named MuStem cells (muscle stem cells), were isolated from healthy dog muscle using a preplating technique. In vitro, MuStem cells displayed a large expansion capacity, an ability to proliferate in suspension, and a multilineage differentiation potential. Phenotypically, they corresponded to early myogenic progenitors and uncommitted cells. When injected in immunosuppressed dystrophic dogs, they contributed to myofiber regeneration, satellite cell replenishment, and dystrophin expression. Importantly, their systemic delivery resulted in long-term dystrophin expression, muscle damage course limitation with an increased regeneration activity and an interstitial expansion restriction, and persisting stabilization of the dog's clinical status. These results demonstrate that MuStem cells could provide an attractive therapeutic avenue for DMD patients.

Goat uterine epithelial cells are susceptible to infection with Caprine Arthritis Encephalitis Virus (CAEV) in vivo.

The aim of this study was to determine, using immunofluorescence and in situ hybridization, whether CAEV is capable of infecting goat uterine epithelial cells in vivo. Five CAEV seropositive goats confirmed as infected using double nested polymerase chain reaction (dnPCR) on leucocytes and on vaginal secretions were used as CAEV positive goats. Five CAEV-free goats were used as controls. Samples from the uterine horn were prepared for dnPCR, in situ hybridization, and immunofluorescence. The results from dnPCR confirmed the presence of CAEV proviral DNA in the uterine horn samples of infected goats whereas no CAEV proviral DNA was detected in samples taken from the uninfected control goats. The in situ hybridization probe was complementary to part of the CAEV gag gene and confirmed the presence of CAEV nucleic acids in uterine samples. The positively staining cells were seen concentrated in the mucosa of the lamina propria of uterine sections. Finally, laser confocal analysis of double p28/cytokeratin immunolabelled transverse sections of CAEV infected goat uterus, demonstrated that the virus was localized in glandular and epithelial cells. This study clearly demonstrates that goat uterine epithelial cells are susceptible to CAEV infection in vivo. This finding could help to further our understanding of the epidemiology of CAEV, and in particular the possibility of vertical transmission.

The Internal Conduit System of the Swine Inverted Lymph Node.

Lymph nodes (LN) are the crossroad where naïve lymphocytes, peripheral antigens and antigen presenting cells contact together in order to mount an adaptive immune response. For this purpose, LN are highly organized convergent hubs of blood and lymphatic vessels that, in the case of B lymphocytes, lead to the B cell follicles. Herein take place the selection and maturation of B cell clones producing high affinity antibodies directed against various antigens. Whereas the knowledge on the murine and human LN distribution systems have reached an exquisite precision those last years, the organization of the antigens and cells circulation into the inverted porcine LN remains poorly described. Using up to date microscopy tools, we described the complex interconnections between afferent lymphatics and blood vessels, perifollicular macrophages, follicular B cells and efferent blood vessels. We observed that afferent lymphatic sinuses presented an asymmetric Lyve-1 expression similar to the one observed in murine LN, whereas specialized perifollicular sinuses connect the main afferent lymphatic sinus to the B cell follicles. Finally, whereas it was long though that mature B cells egress from the inverted LN in the T cell zone through HEV, our observations are in agreement with mature B cells accessing the efferent blood circulation in the efferent, subcapsular area. This understanding of the inverted porcine LN circuitry will allow a more accurate exploration of swine pathogens interactions with the immune cells inside the LN structures. Moreover, the mix between similarities and differences of porcine inverted LN circuitry with mouse and human normal LN shall enable to better apprehend the functions and malfunctions of normal LN from a new perspective.

Quantitative proteomic analysis of dystrophic dog muscle.

Duchenne muscular dystrophy (DMD) is caused by null mutations in the dystrophin gene, leading to progressive and unrelenting muscle loss. Although the genetic basis of DMD is well resolved, the cellular mechanisms associated with the physiopathology remain largely unknown. Increasing evidence suggests that secondary mechanisms, as the alteration of key signaling pathways, may play an important role. In order to identify reliable biomarkers and potential therapeutic targets, and taking advantage of the clinically relevant Golden Retriever Muscular Dystrophy (GRMD) dog model, a proteomic study was performed. Isotope-coded affinity tag (ICAT) profiling was used to compile quantitative changes in protein expression profiles of the vastus lateralis muscles of 4-month old GRMD vs healthy dogs. Interestingly, the set of under-expressed proteins detected appeared primarily composed of metabolic proteins, many of which have been shown to be regulated by the transcriptional peroxisome proliferator-activated receptor-gamma co-activator 1 alpha (PGC-1α). Subsequently, we were able to showed that PGC1-α expression is dramatically reduced in GRMD compared to healthy muscle. Collectively, these results provide novel insights into the molecular pathology of the clinically relevant animal model of DMD, and indicate that defective energy metabolism is a central hallmark of the disease in the canine model.

Neonatal systemic delivery of scAAV9 in rodents and large animals results in gene transfer to RPE cells in the retina.

Systemic delivery of recombinant adeno-associated virus (rAAV) vectors has recently been shown to cross the blood brain barrier in rodents and large animals and to efficiently target cells of the central nervous system. Such approach could be particularly interesting to treat lysosomal storage diseases or neurodegenerative disorders characterized by multiple organs injuries especially neuronal and retinal dysfunctions. However, the ability of rAAV vector to cross the blood retina barrier and to transduce retinal cells after systemic injection has not been precisely determined. In this study, gene transfer was investigated in the retina of neonatal and adult rats after intravenous injection of self-complementary (sc) rAAV serotype 1, 5, 6, 8, and 9 carrying a CMV-driven green fluorescent protein (GFP), by fluorescence fundus photography and histological examination. Neonatal rats injected with scAAV2/9 vector displayed the strongest GFP expression in the retina, within the retinal pigment epithelium (RPE) cells. Retinal tropism of scAAV2/9 vector was further assessed after systemic delivery in large animal models, i.e., dogs and cats. Interestingly, efficient gene transfer was observed in the RPE cells of these two large animal models following neonatal intravenous injection of the vector. The ability of scAAV2/9 to transduce simultaneously neurons in the central nervous system, and RPE cells in the retina, after neonatal systemic delivery, makes this approach potentially interesting for the treatment of infantile neurodegenerative diseases characterized by both neuronal and retinal damages.

Soluble guanylate cyclase chronic stimulation effects on cardiovascular reactivity in cafeteria diet-induced rat model of metabolic syndrome.

Metabolic syndrome is linked to an increased risk of cardiovascular complications by a mechanism involving mainly decreased nitric oxide (NO) bioavailability and impaired NO-soluble guanylate cyclase (sGC)- cyclic guanosine monophosphate (cGMP) signalling (NO-sGC-cGMP). To further develop this scientific point, this study aimed to investigate the effects of long-term treatment with BAY 41-2272 (a sGC stimulator) on cardiovascular reactivity of spontaneously hypertensive rats (SHR) as a model of metabolic syndrome. SHR were randomly divided into 3 groups: control group, cafeteria diet (CD)-fed group and CD-fed group treated daily with BAY 41-2272 (5 mg/kg) by gastric gavage for 12 weeks. In vivo measurements of body weight, abdominal circumference, blood pressure and glucose tolerance test were performed. At the end of the feeding period, ex vivo cumulative concentration-response curves were performed on isolated perfused heart (isoproterenol (0.1 nM - 1 µM)) and thoracic aorta (phenylephrine (1 nM-10 µM), acetylcholine (1 nM-10 µM), and sodium nitroprusside (SNP) (0.1 nM-0.1 µM)). We showed that chronic CD feeding induced abdominal obesity, hypertriglyceridemia, glucose intolerance and exacerbated arterial hypertension in SHR. Compared to control group, CD-fed group showed a decrease in ß-adrenoceptor-induced cardiac inotropy, in coronary perfusion pressure and in aortic contraction to phenylephrine. While relaxing effects of acetylcholine and SNP were unchanged. BAY 41-2272 long-term treatment markedly prevented arterial hypertension development and glucose intolerance, enhanced the α1-adrenoceptor-induced vasoconstriction, and restored cardiac inotropy and coronary vasodilation. These findings suggest that BAY 41-2272 may be a potential novel drug for preventing metabolic and cardiovascular complications of metabolic syndrome.

Confocal spectral microscopy, a non-destructive approach to follow contamination and biofilm formation of mCherry Staphylococcus aureus on solid surfaces.

Methods to test the safety of wood material for hygienically sensitive places are indirect, destructive and limited to incomplete microbial recovery via swabbing, brushing and elution-based techniques. Therefore, we chose mCherry Staphylococcus aureus as a model bacterium for solid and porous surface contamination. Confocal spectral laser microscope (CSLM) was employed to characterize and use the autofluorescence of Sessile oak (Quercus petraea), Douglas fir (Pseudotsuga menziesii) and poplar (Populus euramericana alba L.) wood discs cut into transversal (RT) and tangential (LT) planes. The red fluorescent area occupied by bacteria was differentiated from that of wood, which represented the bacterial quantification, survival and bio-distribution on surfaces from one hour to one week after inoculation. More bacteria were present near the surface on LT face wood as compared to RT and they persisted throughout the study period. Furthermore, this innovative methodology identified that S. aureus formed a dense biofilm on melamine but not on oak wood in similar inoculation and growth conditions. Conclusively, the endogenous fluorescence of materials and the model bacterium permitted direct quantification of surface contamination by using CSLM and it is a promising tool for hygienic safety evaluation.

PTEN contributes to profound PI3K/Akt signaling pathway deregulation in dystrophin-deficient dog muscle.

Duchenne muscular dystrophy is the most common and severe form of muscular dystrophy, and although the genetic basis of this disease is well defined, the overall mechanisms that define its pathogenesis remain obscure. Alterations in individual signaling pathways have been described, but little information is available regarding their putative implications in Duchenne muscular dystrophy pathogenesis. Here, we studied the status of various major signaling pathways in the Golden Retriever muscular dystrophy dog that specifically reproduces the full spectrum of human pathology. Using antibody arrays, we found that Akt1, glycogen synthase kinase-3beta (GSK3beta), 70-kDa ribosomal protein S6 kinase (p70S6K), extracellular signal-regulated kinases 1/2, and p38delta and p38gamma kinases all exhibited decreased phosphorylation in muscle from a 4-month-old animal with Golden Retriever muscular dystrophy, revealing a deep alteration of the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase pathways. Immunohistochemistry analysis revealed the presence of muscle fibers exhibiting a cytosolic accumulation of Akt1, GSK3beta, and phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (PTEN), an enzyme counteracting PI3K-mediated Akt activation. Enzymatic assays established that these alterations in phosphorylation and expression levels were associated with decreased Akt and increased GSK3beta and PTEN activities. PTEN/GSK3beta-positive fibers were also observed in muscle sections from 3- and 36-month-old animals, indicating long-term PI3K/Akt pathway alteration. Collectively, our data suggest that increased PTEN expression and activity play a central role in PI3K/Akt/GSK3beta and p70S6K pathway modulation, which could exacerbate the consequences of dystrophin deficiency.

Efficient intrathymic gene transfer following in situ administration of a rAAV serotype 8 vector in mice and nonhuman primates.

The thymus is the primary site of T-cell development and plays a key role in the induction of self-tolerance. We previously showed that the intrathymic (i.t.) injection of a transgene-expressing lentiviral vector (LV) in mice can result in the correction of a T cell-specific genetic defect. Nevertheless, the efficiency of thymocyte transduction did not exceed 0.1-0.3% and we were unable to detect any thymus transduction in macaques. As such, we initiated studies to assess the capacity of recombinant adeno-associated virus (rAAV) vectors to transduce murine and primate thymic cells. In vivo administration of AAV serotype 2-derived single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors pseudotyped with capsid proteins of serotypes 1, 2, 4, 5, and 8 demonstrated that murine thymus transduction was significantly enhanced by scAAV2/8. Transgene expression was detected in 5% of thymocytes and, notably, transduced cells represented 1% of peripheral T lymphocytes. Moreover, i.t. administration of scAAV2/8 particles in macaques, by endoscopic-mediated guidance, resulted in significant gene transfer. Thus, in healthy animals, where thymic gene transfer does not provide a selective advantage, scAAV2/8 is a unique tool promoting the in situ transduction of thymocytes with the subsequent export of gene-modified lymphocytes to the periphery.