Ly49D engagement on T lymphocytes induces TCR-independent activation and CD8 effector functions that control tumor growth.

Recent data showing expression of activating NK receptors (NKR) by conventional T lymphocytes raise the question of their role in the triggering of TCR-independent responses that could be damaging for the host. Transgenic mice expressing the activating receptor Ly49D/DAP12 offer the opportunity to better understand the relevance of ITAM signaling in the biology of T cells. In vitro experiments showed that Ly49D engagement on T lymphocytes by a cognate MHC class I ligand expressed by Chinese hamster ovary (CHO) cells or by specific Ab triggered cellular activation of both CD4 and CD8 populations with modulation of activation markers and cytokine production. The forced expression of the ITAM signaling chain DAP12 is mandatory for Ly49D-transgenic T cell activation. In addition, Ly49D stimulation induced T lymphocyte proliferation, which was much stronger for CD8 T cells. Phenotypic analysis of anti-Ly49D-stimulated CD8 T cells and their ability to produce high levels of IFN-gamma and to kill target cells indicate that Ly49D ligation generates effector cytotoxic CD8 T cells. Ly49D engagement by itself also triggered cytotoxic activity of activated CD8 T cells. Adoptive transfer experiments confirmed that Ly49D-transgenic CD8 T cells are able to control growth of CHO tumor cells or RMA cells transfected with Hm1-C4, the Ly49D ligand normally expressed by CHO. In conclusion, Ly49D engagement on T cells leads to T cell activation and to a full range of TCR-independent effector functions of CD8 T cells.

T cell receptor beta chain gene rearrangements and V beta gene usage in horse cytochrome c-specific T cell hybridomas.

Comparison of T cell receptor alpha and beta-chain genes in murine major histocompatibility complex (MHC) class I and class II-restricted T cell clones and hybridomas recognizing different antigens indicates that no simple correlation exists between the observed antigen/MHC specificity and the expression of certain alpha and beta-chain heterodimers. We have attempted to establish a possible correlation by analyzing T cell receptor beta chain gene rearrangements and V beta gene usage in five T cell hybridomas with identical antigen/MHC specificity and another hybridoma recognizing a different antigenic determinant in association with the same restriction molecule. We report here that in each of the five clones a uniquely rearranged beta chain gene is expressed in combination with at least two different V beta gene segments. The presence of the differently rearranged T cell receptor beta chain genes correlated with the finding of distinct fine specificity pattern of antigen recognition in each of the hybridomas. Interestingly, two hybridomas specific for different epitopes showed identical beta chain D-J rearrangements indicating that the differences might be encoded by the alpha chain gene or/and the V beta gene element.

Human muscular fetal cells: a potential cell source for muscular therapies.

Myoblast transfer therapy has been extensively studied for a wide range of clinical applications, such as tissue engineering for muscular loss, cardiac surgery or Duchenne Muscular Dystrophy treatment. However, this approach has been hindered by numerous limitations, including early myoblast death after injection and specific immune response after transplantation with allogenic cells. Different cell sources have been analyzed to overcome some of these limitations. The object of our study was to investigate the growth potential, characterization and integration in vivo of human primary fetal skeletal muscle cells. These data together show the potential for the creation of a cell bank to be used as a cell source for muscle cell therapy and tissue engineering. For this purpose, we developed primary muscular cell cultures from biopsies of human male thigh muscle from a 16-week-old fetus and from donors of 13 and 30 years old. We show that fetal myogenic cells can be successfully isolated and expanded in vitro from human fetal muscle biopsies, and that fetal cells have higher growth capacities when compared to young and adult cells. We confirm lineage specificity by comparing fetal muscle cells to fetal skin and bone cells in vitro by immunohistochemistry with desmin and 5.1 H11 antibodies. For the feasibility of the cell bank, we ensured that fetal muscle cells retained intrinsic characteristics after 5 years cryopreservation. Finally, human fetal muscle cells marked with PKH26 were injected in normal C57BL/6 mice and were found to be present up to 4 days. In conclusion we estimate that a human fetal skeletal muscle cell bank can be created for potential muscle cell therapy and tissue engineering.

Mass conservative three-dimensional water tracer distribution from Markov chain Monte Carlo inversion of time-lapse ground-penetrating radar data

Time-lapse geophysical measurements are widely used to monitor the movement of water and solutes through the subsurface. Yet commonly used deterministic least squares inversions typically suffer from relatively poor mass recovery, spread overestimation, and limited ability to appropriately estimate nonlinear model uncertainty. We describe herein a novel inversion methodology designed to reconstruct the three-dimensional distribution of a tracer anomaly from geophysical data and provide consistent uncertainty estimates using Markov chain Monte Carlo simulation. Posterior sampling is made tractable by using a lower-dimensional model space related both to the Legendre moments of the plume and to predefined morphological constraints. Benchmark results using cross-hole ground-penetrating radar travel times measurements during two synthetic water tracer application experiments involving increasingly complex plume geometries show that the proposed method not only conserves mass but also provides better estimates of plume morphology and posterior model uncertainty than deterministic inversion results.

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.

Gene transfer into the airway epithelium of animals by targeting the polymeric immunoglobulin receptor.

Genes of interest can be targeted specifically to respiratory epithelial cells in intact animals with high efficiency by exploiting the receptor-mediated endocytosis of the polymeric immunoglobulin receptor. A DNA carrier, consisting of the Fab portion of polyclonal antibodies raised against rat secretory component covalently linked to poly-L-lysine, was used to introduce plasmids containing different reporter genes into airway epithelial cells in vivo. We observed significant levels of luciferase enzyme activity in protein extracts from the liver and lung, achieving maximum values of 13,795 +/- 4,431 and 346,954 +/- 199,120 integrated light units (ILU) per milligram of protein extract, respectively. No luciferase activity was detected in spleen or heart, which do not express the receptor. Transfections using complexes consisting of an irrelevant plasmid (pCMV lacZ) bound to the bona fide carrier or the expression plasmid (pGEMluc) bound to a carrier based on an irrelevant Fab fragment resulted in background levels of luciferase activity in all tissues examined. Thus, only tissues that contain cells bearing the polymeric immunoglobulin receptor are transfected, and transfection cannot be attributed to the nonspecific uptake of an irrelevant carrier-DNA complex. Specific mRNA from the luciferase gene was also detected in the lungs of transfected animals. To determine which cells in the lungs are transfected by this method, DNA complexes were prepared containing expression plasmids with genes encoding the bacterial beta-galactosidase or the human interleukin 2 receptor. Expression of these genes was localized to the surface epithelium of the airways and the submucosal glands, and not the bronchioles and alveoli. Receptor-mediated endocytosis can be used to introduce functional genes into the respiratory epithelium of rats, and may be a useful technique for gene therapy targeting the lung.

Respiratory chain dysfunction associated with multiple mitochondrial DNA deletions in antiretroviral therapy-related lipodystrophy.

Highly-active antiretroviral therapy (HAART) can induce a characteristic lipodystrophy syndrome characterized by peripheral fat wasting and central adiposity, usually associated with hyperlipidaemia and insulin resistance [1,2]. Indirect data have led some authors to propose that mitochondrial dysfunction could play a role in this syndrome [3,4].To date, as recently outlined by Kakuda et al. [5] in this journal, HIV-infected patients developing lipodystrophy have not been studied for mitochondrial changes or respiratory chain capacity...

Non integrative lentiviral vactors for gene transfer in the retina

Purpose:Lentiviral vectors are among the most efficient gene transfer tools for both dividing and non dividing cells, including pigmented epithelial cells of the retina. One of the latest developments in the field, which represents a significant advance in biosafety, consists in the use of non integrative lentiviral vectors (NILVs). These newly described tools were already shown to be efficient in various tissues, such as the retina. They allow prolonged transgene expression as long as the transduced cells do not divide or divide slowly. However, they were also shown to induce transgene expression less efficiently than their integrative counterparts. Further investigations are thus needed to improve their potential. To this aim, different strategies are under evaluation. In this study, we focused on using different integrase mutations. Methods:We considered different integrase mutations, including modifications in the catalytic site and in the C-terminal domain of the enzyme. Lentiviral vectors bearing these mutant integrases and allowing expression of various transgenes were produced and characterized in vitro and in vivo. In particular, we evaluated their transgene expression capability. Influence of integrase mutation on the residual integration activity was also investigated. Results:In line with the fact that the lentiviral integrase is involved in several steps of the replication cycle of lentiviruses, we observed that integrase mutations can modify lentiviral vector features, resulting in different transduction efficiencies as well as modulation of the integration activity. Conclusions:NILVs appear as suitable tools for gene transfer in the retina, particularly to transduce RPE cells. They can be advantageously used, for instance, to develop neuroprotective strategies aimed at rescuing photoreceptors from death in various retinal diseases.

Encapsulation of packaging cell line results in successful retroviral-mediated transfer of a suicide gene in vivo in an experimental model of glioblastoma.

AIMS: Retroviral-mediated gene therapy has been proposed as a primary or adjuvant treatment for advanced cancer, because retroviruses selectively infect dividing cells. Efficacy of retroviral-mediated gene transfer, however, is limited in vivo. Although packaging cell lines can produce viral vectors continuously, such allo- or xenogeneic cells are normally rejected when used in vivo. Encapsulation using microporous membranes can protect the packaging cells from rejection. In this study, we used an encapsulated murine packaging cell line to test the effects of in situ delivery of a retrovirus bearing the herpes simplex virus thymidine kinase suicide gene in a rat model of orthotopic glioblastoma. MATERIALS AND METHODS: To test gene transfer in vitro, encapsulated murine psi2-VIK packaging cells were co-cultured with baby hamster kidney (BHK) cells, and the percentage of transfected BHK cells was determined. For in vivo experiments, orthotopic C6 glioblastomas were established in Wistar rats. Capsules containing psi2-VIK cells were stereotaxically implanted into these tumours and the animals were treated with ganciclovir (GCV). Tumours were harvested 14 days after initiation of GCV therapy for morphometric analysis. RESULTS: Encapsulation of psi2-VIK cells increased transfection rates of BHK target cells significantly in vitro compared to psi2-VIK conditioned medium (3 x 10(6) vs 2.3 x 10(4) cells; P<0.001). In vivo treatment with encapsulated packaging cells resulted in 3% to 5% of C6 tumour cells transduced and 45% of tumour volume replaced by necrosis after GCV (P<0.01 compared to controls). CONCLUSION: In this experimental model of glioblastoma, encapsulation of a xenogeneic packaging cell line increased half-life and transduction efficacy of retrovirus-mediated gene transfer and caused significant tumour necrosis.

Localized amyloid light-chain amyloidosis and extramedullary plasmacytoma of the mitral valve.

An unusual case of localized amyloid light-chain (AL) amyloidosis and extramedullary plasmacytoma of the mitral valve is described. The worsening of a mitral regurgitation led to investigations and surgery. The valve presented marked distortion and thickening by type AL amyloid associated with a monotypic CD138+ immunoglobulin lambda plasma cell proliferation. Systemic staging showed a normal bone marrow and no evidence of amyloid deposition in other localizations. The patient's outcome after mitral valve replacement was excellent. To our knowledge, this is the first description of a localized AL amyloidosis as well as of a primary extramedullary plasmacytoma of the mitral valve.

The interplay between relatedness and horizontal gene transfer drives the evolution of plasmid-carried public goods.

Plasmids carry a wide range of genes that are often involved in bacterial social behaviour. The question of why such genes are frequently mobile has received increasing attention. Here, we use an explicit population genetic approach to model the evolution of plasmid-borne bacterial public goods production. Our findings highlight the importance of both transmission and relatedness as factors driving the evolution of plasmid-borne public goods production. We partition the effects of plasmid transfer of social traits into those of infectivity and the effect of increased relatedness. Our results demonstrate that, owing to its effect on relatedness, plasmid mobility increases the invasion and stability of public goods, in a way not seen in individually beneficial traits. In addition, we show that plasmid transfer increases relatedness when public goods production is rare but this effect declines when production is common, with both scenarios leading to an increase in the frequency of plasmid-borne public goods. Plasmids remain important vectors for the spread of social genes involved in bacterial virulence thus an understanding of their dynamics is highly relevant from a public health perspective.

Efficient energy transfer from Si clusters to Er3+ in complex silicate glasses

We present an extensive study of the structural and optical emission properties in aluminum silicates and soda-lime silicates codoped with Si nanoclusters (Si-nc) and Er. Si excess of 5 and 15¿at.¿% and Er concentrations ranging from 2×1019 up to 6×1020¿cm¿3 were introduced by ion implantation. Thermal treatments at different temperatures were carried out before and after Er implantation. Structural characterization of the resulting structures was performed to obtain the layer composition and the size distribution of Si clusters. A comprehensive study has been carried out of the light emission as a function of the matrix characteristics, Si and Er contents, excitation wavelength, and power. Er emission at 1540¿nm has been detected in all coimplanted glasses, with similar intensities. We estimated lifetimes ranging from 2.5¿to¿12¿ms (depending on the Er dose and Si excess) and an effective excitation cross section of about 1×10¿17¿cm2 at low fluxes that decreases at high pump power. By quantifying the amount of Er ions excited through Si-nc we find a fraction of 10% of the total Er concentration. Upconversion coefficients of about 3×10¿18¿cm¿3¿s¿1 have been found for soda-lime glasses and one order of magnitude lower in aluminum silicates.

Hit parade for adoptive cell transfer therapy: the best T cells for superior clinical responses.

Adoptive cell transfer (ACT) of T cells has great clinical potential, but the numerous variables of this therapy make choices difficult. A new study takes advantage of a novel technology for characterizing the T-cell responses of patients. If applied systematically, this approach may identify biomedical correlates of protection, thereby supporting treatment optimization.

Transfer insensitive labeling technique (TILT): application to multislice functional perfusion imaging.

Cerebral blood flow can be studied in a multislice mode with a recently proposed perfusion sequence using inversion of water spins as an endogenous tracer without magnetization transfer artifacts. The magnetization transfer insensitive labeling technique (TILT) has been used for mapping blood flow changes at a microvascular level under motor activation in a multislice mode. In TILT, perfusion mapping is achieved by subtraction of a perfusion-sensitized image from a control image. Perfusion weighting is accomplished by proximal blood labeling using two 90 degrees radiofrequency excitation pulses. For control preparation the labeling pulses are modified such that they have no net effect on blood water magnetization. The percentage of blood flow change, as well as its spatial extent, has been studied in single and multislice modes with varying delays between labeling and imaging. The average perfusion signal change due to activation was 36.9 +/- 9.1% in the single-slice experiments and 38.1 +/- 7.9% in the multislice experiments. The volume of activated brain areas amounted to 1.51 +/- 0.95 cm3 in the contralateral primary motor (M1) area, 0.90 +/- 0.72 cc in the ipsilateral M1 area, 1.27 +/- 0.39 cm3 in the contralateral and 1.42 +/- 0.75 cm3 in the ipsilateral premotor areas, and 0.71 +/- 0.19 cm3 in the supplementary motor area.