T staging esophageal tumors with x rays.

With histopathology results typically taking several days, the ability to stage tumors during interventions could provide a step change in various cancer interventions. X-ray technology has advanced significantly in recent years with the introduction of phase-based imaging methods. These have been adapted for use in standard labs rather than specialized facilities such as synchrotrons, and approaches that enable fast 3D scans with conventional x-ray sources have been developed. This opens the possibility to produce 3D images with enhanced soft tissue contrast at a level of detail comparable to histopathology, in times sufficiently short to be compatible with use during surgical interventions. In this paper we discuss the application of one such approach to human esophagi obtained from esophagectomy interventions. We demonstrate that the image quality is sufficiently high to enable tumor T staging based on the x-ray datasets alone. Alongside detection of involved margins with potentially life-saving implications, staging tumors intra-operatively has the potential to change patient pathways, facilitating optimization of therapeutic interventions during the procedure itself. Besides a prospective intra-operative use, the availability of high-quality 3D images of entire esophageal tumors can support histopathological characterization, from enabling "right slice first time" approaches to understanding the histopathology in the full 3D context of the surrounding tumor environment.

The effect of a variable focal spot size on the contrast channels retrieved in edge-illumination X-ray phase contrast imaging.

Multi-modal X-ray imaging allows the extraction of phase and dark-field (or "Ultra-small Angle Scatter") images alongside conventional attenuation ones. Recently, scan-based systems using conventional sources that can simultaneously output the above three images on relatively large-size objects have been developed by various groups. One limitation is the need for some degree of spatial coherence, achieved either through the use of microfocal sources, or by placing an absorption grating in front of an extended source. Both these solutions limit the amount of flux available for imaging, with the latter also leading to a more complex setup with additional alignment requirements. Edge-illumination partly overcomes this as it was proven to work with focal spots of up to 100 micron. While high-flux, 100 micron focal spot sources do exist, their comparatively large footprint and high cost can be obstacles to widespread translation. A simple solution consists in placing a single slit in front of a large focal spot source. We used a tunable slit to study the system performance at various effective focal spot sizes, by extracting transmission, phase and dark-field images of the same specimens for a range of slit widths. We show that consistent, repeatable results are obtained for varying X-ray statistics and effective focal spot sizes. As the slit width is increased, the expected reduction in the raw differential phase peaks is observed, compensated for in the retrieval process by a broadened sensitivity function. This leads to the same values being correctly retrieved, but with a slightly larger error bar i.e. a reduction in phase sensitivity. Concurrently, a slight increase in the dark-field signal is also observed.

Enhanced detection of threat materials by dark-field x-ray imaging combined with deep neural networks.

X-ray imaging has been boosted by the introduction of phase-based methods. Detail visibility is enhanced in phase contrast images, and dark-field images are sensitive to inhomogeneities on a length scale below the system's spatial resolution. Here we show that dark-field creates a texture which is characteristic of the imaged material, and that its combination with conventional attenuation leads to an improved discrimination of threat materials. We show that remaining ambiguities can be resolved by exploiting the different energy dependence of the dark-field and attenuation signals. Furthermore, we demonstrate that the dark-field texture is well-suited for identification through machine learning approaches through two proof-of-concept studies. In both cases, application of the same approaches to datasets from which the dark-field images were removed led to a clear degradation in performance. While the small scale of these studies means further research is required, results indicate potential for a combined use of dark-field and deep neural networks in security applications and beyond.

A comparison of the physiological consequences of head-loading and back-loading for African and European women.

The aim is to quantify the physiological cost of head-load carriage and to examine the 'free ride' hypothesis for head-load carriage in groups of women differing in their experience of head-loading. Twenty-four Xhosa women [13 experienced head-loaders (EXP), 11 with no experience of head-loading (NON)] attempted to carry loads of up to 70% of body mass on both their heads and backs whilst walking on a treadmill at a self-selected walking speed. Expired air was collected throughout. In a second study nine women, members of the British Territorial Army, carried similar loads, again at a self-selected speed. Maximum load carried was greater for the back than the head (54.7 +/- 15.1 vs. 40.8 +/- 13.2% BM, P < 0.0005). Considering study one, head-loading required a greater oxygen rate than back-loading (10.1 +/- 2.6 vs. 8.8 +/- 2.3 ml kg bodymass(-1) min(-1), P = 0.043, for loads 10-25% BM) regardless of previous head-loading experience (P = 0.333). Percentage changes in oxygen consumption associated with head-loading were greater than the proportional load added in both studies but were smaller than the added load for the lighter loads carried on the back in study 1. All other physiological variables were consistent with changes in oxygen consumption. The data provides no support for the 'free ride' hypothesis for head-loading although there is some evidence of energy saving mechanisms for back-loading at low speed/load combinations. Investigating the large individual variation in response may help in identifying combinations of factors that contribute to improved economy.

Normal myogenic cells from newborn mice restore normal histology to degenerating muscles of the mdx mouse.

Dystrophin deficiency in skeletal muscle of the x-linked dystrophic (mdx) mouse can be partially remedied by implantation of normal muscle precursor cells (mpc) (Partridge, T. A., J. E. Morgan, G. R. Coulton, E. P. Hoffman, and L. M. Kunkel. 1989. Nature (Lond.). 337:176-179). However, it is difficult to determine whether this biochemical "rescue" results in any improvement in the structure or function of the treated muscle, because the vigorous regeneration of mdx muscle more than compensates for the degeneration (Coulton, G. R., N. A. Curtin, J. E. Morgan, and T. A. Partridge. 1988. Neuropathol. Appl. Neurobiol. 14:299-314). By using x-ray irradiation to prevent mpc proliferation, it is possible to study loss of mdx muscle fibers without the complicating effect of simultaneous fiber regeneration. Thus, improvements in fiber survival resulting from any potential therapy can be detected easily (Wakeford, S., D. J. Watt, and T. A. Patridge. 1990. Muscle & Nerve.) Here, we have implanted normal mpc, obtained from newborn mice, into such preirradiated mdx muscles, finding that it is far more extensively permeated and replaced by implanted mpc than is nonirradiated mdx muscle; this is evident both from analysis of glucose-6-phosphate isomerase isoenzyme markers and from immunoblots and immunostaining of dystrophin in the treated muscles. Incorporation of normal mpc markedly reduces the loss of muscle fibers and the deterioration of muscle structure which otherwise occurs in irradiated mdx muscles. Surprisingly, the regenerated fibers are largely peripherally nucleated, whereas regenerated mouse skeletal muscle fibers are normally centrally nucleated. We attribute this regeneration of apparently normal muscle to the tendency of newborn mouse mpc to recapitulate their neonatal ontogeny, even when grafted into 3-wk-old degenerating muscle.

Dynamics of myoblast transplantation reveal a discrete minority of precursors with stem cell-like properties as the myogenic source.

Myoblasts, the precursors of skeletal muscle fibers, can be induced to withdraw from the cell cycle and differentiate in vitro. Recent studies have also identified undifferentiated subpopulations that can self-renew and generate myogenic cells (Baroffio, A., M. Hamann, L. Bernheim, M.-L. Bochaton-Pillat, G. Gabbiani, and C.R. Bader. 1996. Differentiation. 60:47-57; Yoshida, N., S. Yoshida, K. Koishi, K. Masuda, and Y. Nabeshima. 1998. J. Cell Sci. 111:769-779). Cultured myoblasts can also differentiate and contribute to repair and new muscle formation in vivo, a capacity exploited in attempts to develop myoblast transplantation (MT) for genetic modification of adult muscle. Our studies of the dynamics of MT demonstrate that cultures of myoblasts contain distinct subpopulations defined by their behavior in vitro and divergent responses to grafting. By comparing a genomic and a semiconserved marker, we have followed the fate of myoblasts transplanted into muscles of dystrophic mice, finding that the majority of the grafted cells quickly die and only a minority are responsible for new muscle formation. This minority is behaviorally distinct, slowly dividing in tissue culture, but rapidly proliferative after grafting, suggesting a subpopulation with stem cell-like characteristics.

Massive idiosyncratic exon skipping corrects the nonsense mutation in dystrophic mouse muscle and produces functional revertant fibers by clonal expansion.

Conventionally, nonsense mutations within a gene preclude synthesis of a full-length functional protein. Obviation of such a blockage is seen in the mdx mouse, where despite a nonsense mutation in exon 23 of the dystrophin gene, occasional so-called revertant muscle fibers are seen to contain near-normal levels of its protein product. Here, we show that reversion of dystrophin expression in mdx mice muscle involves unprecedented massive loss of up to 30 exons. We detected several alternatively processed transcripts that could account for some of the revertant dystrophins and could not detect genomic deletion from the region commonly skipped in revertant dystrophin. This, together with exon skipping in two noncontiguous regions, favors aberrant splicing as the mechanism for the restoration of dystrophin, but is hard to reconcile with the clonal idiosyncrasy of revertant dystrophins. Revertant dystrophins retain functional domains and mediate plasmalemmal assembly of the dystrophin-associated glycoprotein complex. Physiological function of revertant fibers is demonstrated by the clonal growth of revertant clusters with age, suggesting that revertant dystrophin could be used as a guide to the construction of dystrophin expression vectors for individual gene therapy. The dystrophin gene in the mdx mouse provides a favored system for study of exon skipping associated with nonsense mutations.

Expression of CD34 and Myf5 defines the majority of quiescent adult skeletal muscle satellite cells.

Skeletal muscle is one of a several adult post-mitotic tissues that retain the capacity to regenerate. This relies on a population of quiescent precursors, termed satellite cells. Here we describe two novel markers of quiescent satellite cells: CD34, an established marker of hematopoietic stem cells, and Myf5, the earliest marker of myogenic commitment. CD34(+ve) myoblasts can be detected in proliferating C2C12 cultures. In differentiating cultures, CD34(+ve) cells do not fuse into myotubes, nor express MyoD. Using isolated myofibers as a model of synchronous precursor cell activation, we show that quiescent satellite cells express CD34. An early feature of their activation is alternate splicing followed by complete transcriptional shutdown of CD34. This data implicates CD34 in the maintenance of satellite cell quiescence. In heterozygous Myf5(nlacZ/+) mice, all CD34(+ve) satellite cells also express beta-galactosidase, a marker of activation of Myf5, showing that quiescent satellite cells are committed to myogenesis. All such cells are positive for the accepted satellite cell marker, M-cadherin. We also show that satellite cells can be identified on isolated myofibers of the myosin light chain 3F-nlacZ-2E mouse as those that do not express the transgene. The numbers of satellite cells detected in this way are significantly greater than those identified by the other three markers. We conclude that the expression of CD34, Myf5, and M-cadherin defines quiescent, committed precursors and speculate that the CD34(-ve), Myf5(-ve) minority may be involved in maintaining the lineage-committed majority.

Optimizing exon skipping therapies for DMD.

Exon skipping is one of the more promising therapeutic options for Duchenne Muscular Dystrophy (DMD). The idea is to use antisense oligonucleotides to splice out selected exons from the pre-mRNA, at or next to the mutation site, so as to generate a translatable transcript from the mutant dystrophin gene. In principle, the majority of DMD mutations can be rescued by targeting selected exons. Recent developments of antisense oligonucleotides (AOs) such as 2O-methylated antisense oligonucleotides (2OMeAOs) or phosphorodiamidate morpholino oligomers (morpholinos, PMOs) have made it possible to restore dystrophin expression body-wide in dystrophic mice and dystrophic dogs by single or multi-exon skipping with no obvious side-effect. Since such treatment would, in many cases, require bespoke design of AOs, it is important to demonstrate treatment of a variety of mutations in dystrophic animals. In-frame deletion patterns usually result in a mix of Duchenne and milder Becker Muscular Dystrophy (BMD), but the ratio of Duchenne to Becker varies between patterns, and this provides useful information for selection of the exons that might most profitably be targeted. This review summarizes recent progress in exon skipping therapy and discusses future strategies.

Viruses transfer the antiviral second messenger cGAMP between cells.

Cyclic GMP-AMP synthase (cGAS) detects cytosolic DNA during virus infection and induces an antiviral state. cGAS signals by synthesis of a second messenger, cyclic GMP-AMP (cGAMP), which activates stimulator of interferon genes (STING). We show that cGAMP is incorporated into viral particles, including lentivirus and herpesvirus virions, when these are produced in cGAS-expressing cells. Virions transferred cGAMP to newly infected cells and triggered a STING-dependent antiviral program. These effects were independent of exosomes and viral nucleic acids. Our results reveal a way by which a signal for innate immunity is transferred between cells, potentially accelerating and broadening antiviral responses. Moreover, infection of dendritic cells with cGAMP-loaded lentiviruses enhanced their activation. Loading viral vectors with cGAMP therefore holds promise for vaccine development.

Human muscle precursor cell regeneration in the mouse host is enhanced by growth factors.

The aim of this study was to optimize human muscle formation in vivo from implanted human muscle precursor cells. We transplanted donor muscle precursor cells (MPCs) prepared from postnatal or fetal human muscle into immunodeficient host mice and showed that irradiation of host muscle significantly enhanced muscle formation by donor cells. The amount of donor muscle formed in cryodamaged host muscle was increased by exposure of donor cells to growth factors before their implantation into injured host muscle. Insulin-like growth factor type I (IGF-I) significantly increased the amount of muscle formed by postnatal human muscle cells, but not by fetal human MPCs. However, treatment of fetal muscle cells with IGF-I, in combination with basic fibroblast growth factor and plasmin, significantly increased the amount of donor muscle formed. In vivo, human MPCs formed mosaic human-mouse muscle fibers, in which each human myonucleus was associated with a zone of human sarcolemmal protein spectrin.

A new immunodeficient mouse model for human myoblast transplantation.

Design of efficient transplantation strategies for myoblast-based gene therapies in humans requires animal models in which xenografts are tolerated for long periods of time. In addition, such recipients should be able to withstand pretransplantation manipulations for enhancement of graft growth. Here we report that a newly developed immunodeficient mouse carrying two known mutations (the recombinase activating gene 2, RAG2, and the common cytokine receptor gamma, gammac) is a candidate fulfilling these requirements. Skeletal muscles from RAG2(-/-)/gammac(-/-) double mutant mice recover normally after myotoxin application or cryolesion, procedures commonly used to induce regeneration and improve transplantation efficiency. Well-differentiated donor-derived muscle tissue could be detected up to 9 weeks after transplantation of human myoblasts into RAG2(-/-)/gammac(-/-) muscles. These results suggest that the RAG2(-/-)/gammac(-/-) mouse model will provide new opportunities for human muscle research.

Viral gene delivery to skeletal muscle: insights on maturation-dependent loss of fiber infectivity for adenovirus and herpes simplex type 1 viral vectors.

The mechanisms causing age-dependent loss of muscle fiber infectivity observed in vivo for both adenoviral (Ad) and herpes simplex virus type 1 (HSV-1) gene delivery vectors remain poorly understood. Here we investigate the possible bases for this phenomenon using the novel application of enzymatically isolated, viable, single muscle fibers. We show that maturation-dependent loss of fiber infectivity is recapitulated in single fibers, and, thus, is not solely due to host immune response. Using localized irradiation of muscle in vivo, we show data suggesting that Ad infectivity of differentiated myofibers depends, at least in part, on myoblasts to mediate fiber transduction. On the other hand, infection of single fibers by HSV-1 is not affected by irradiation. Using confocal microscopy, we show that the basal lamina of myogenic cells efficiently infected by HSV-1 is structurally less organized than that of fibers resistant to infection by HSV-1. As well, we show that single myofibers isolated from adult, basal lamina-defective mice (merosin-deficient, dy/dy) are at least 10-fold more susceptible to infection by HSV-1 than are myofibers isolated from control mice. Together, these observations support the hypothesis that the basal lamina acts as a physical barrier to HSV-1 infection of mature muscle.

Allografts of muscle precursor cells persist in the non-tolerized host.

Implantation of normal muscle precursor cells into myopathic fibres to alleviate recessively inherited diseases of skeletal muscle has received much attention since the discovery of a defective or deficient gene coding for the protein dystrophin in the Duchenne and Becker forms of muscular dystrophy. Therapeutic allografting of cells would require some means of preventing their immune rejection. Here we have allografted muscle into the non-tolerant and non-immunosuppressed murine host. Precursor cells introduced in the form of a single cell suspension survive for prolonged periods post-implantation. Allografts of minced muscle often failed to survive, even though host and donor were compatible at the major histocompatibility locus. Differences at minor loci may well have contributed to such rejection. Where allografted tissue was rejected, there was a decrease in the amount of surviving host muscle at the graft site, an important observation in terms of the therapeutic implantation of cells.

A dual-marker system for quantitative studies of myoblast transplantation in the mouse.

BACKGROUND: Myoblast transplantation (MT) is a potential approach for gene transfer into skeletal muscle, the efficiency of which depends upon the number of copies of donor genome incorporated into the host tissue. We have developed a system for quantitative studies of MT that measures amounts of donor-derived genome in host muscles and estimates the contributions of donor cell survival and proliferation in vivo. METHODS: [14C]thymidine-labeled, male myoblasts were transplanted into female muscles, providing two donor cell markers, Y chromosome and [14C]. The markers were measured in muscle extracts by slot blotting and scintillation counting, respectively. RESULTS: In each extract, the amount of Y chromosome was used to quantify donor-derived genome, whereas the radiolabel provided an estimate of cell survival. Furthermore, the different modes of inheritance of the markers meant that proliferation of surviving donor cells was detected as a change in marker ratio. CONCLUSIONS: This system provides a method for assessing potential improvements of MT.

Cyclosporin A as a means of preventing rejection of skeletal muscle allografts in mice.

Isografts and allografts of skeletal muscle inserted into the limbs of mice initially degenerate. After some 5 to 8 days newly formed myotubes appear in the graft which develop into mature muscle fibers. In nontolerant hosts allografts are rejected between the 10th and 12th days. In mice treated with cyclosporin A, this effect persists for some 12 days after the end of treatment. Isoenzyme marker studies indicate that the regenerated graft is composed of both host and donor tissue. Donor isoenzyme does not persist when grafts are rejected.

A new blocking method for application of murine monoclonal antibody to mouse tissue sections.

Antigen detection with primary antibody of the same species as the test tissue is complicated by high levels of background staining when indirect immunohistochemical detection methods are used. This severely limits the use of murine monoclonal antibodies on tissues of the mouse, the most widely used experimental model system; no method for blocking this is fully satisfactory. Here we show that background staining encountered in this system results largely from the binding of secondary antibodies via both Fc and Fab to endogenous immunoglobulins and other tissue components. A simple and efficient blocking strategy was established, employing papain-digested whole fragments of unlabeled secondary anti-mouse Igs enriched with Fc fragment of the same Igs. We have used this method to visualize dystrophin, an antigen expressed at low level, in revertant fibers of mdx mouse by both immunoperoxidase and immunofluorescence methods. In combination with the use of a biotin-streptavidin immunohistochemical detection protocol with biotinylated anti-mouse F(ab')2 as second layer, we eliminated the heavy background in this system and achieved strong signal amplification to demonstrate the specific antigen clearly. Double labeling with one mouse antibody and one antibody from another species was performed without signal interference. This principle can be adapted for wider applications, such as antibodies of other species on homologous tissues and perhaps where high background is found with heterologous antibodies. (J Histochem Cytochem 46:977-983, 1998)

Representative sample of rheumatoid synovium: a morphometric study.

The synovium from 11 patients with rheumatoid arthritis, who were undergoing joint surgery, was assessed using histological and morphometric techniques. Histological examination confirmed previous reports that the intensity of the cellular reaction varied throughout the synovium, and the morphometric method reflected this variability sensitively. The method was shown to be reproducible and allowed areas of similar cellular density to be defined. From these defined areas a total of 2.5 mm2 of synovium equivalent to 12 fields at x250 required analysis to reflect the variation in the cellular reaction. It would be feasible to collect this amount of material using an arthroscope.