We used the model nematode Caenorhabditis elegans infected with the human pathogen Staphylococcus aureus to identify components of epithelial immunity. Transcriptional profiling and reverse genetic analysis revealed that mutation of the C. elegans beta-catenin homolog bar-1 or the downstream homeobo

We used the model nematode Caenorhabditis elegans infected with the human pathogen Staphylococcus aureus to identify components of epithelial immunity. Transcriptional profiling and reverse genetic analysis revealed that mutation of the C. elegans beta-catenin homolog bar-1 or the downstream homeobox gene egl-5 results in a defective response and hypersensitivity to S. aureus infection. Epistasis analysis showed that bar-1 and egl-5 function in parallel to previously described C. elegans immune-response pathways. Overexpression of human homologs of egl-5 modulated NF-kappaB-dependent TLR2 signaling in epithelial cells. These data suggest that beta-catenin and homeobox genes play an important and conserved role in innate immune defense.

The genetically tractable model organism Caenorhabditis elegans was first used to model bacterial virulence in vivo a decade ago. Since then, great strides have been made in identifying the host response pathways that are involved in its defence against infection. Strikingly, C. elegans seems to det

The genetically tractable model organism Caenorhabditis elegans was first used to model bacterial virulence in vivo a decade ago. Since then, great strides have been made in identifying the host response pathways that are involved in its defence against infection. Strikingly, C. elegans seems to detect, and respond to, infection without the involvement of its homologue of Toll-like receptors, in contrast to the well-established role for these proteins in innate immunity in mammals. What, therefore, do we know about host defence mechanisms in C. elegans and what can they tell us about innate immunity in higher organisms?

The implications of priming experiments for a structural theory of the mental lexicon depend critically on the development of an adequate theory of the mechanics of priming. Masked priming techniques may simplify this task, since consciously perceived relationships play no role in masked priming. Th

The implications of priming experiments for a structural theory of the mental lexicon depend critically on the development of an adequate theory of the mechanics of priming. Masked priming techniques may simplify this task, since consciously perceived relationships play no role in masked priming. The implications of adopting an activation-based approach to morphological priming is discussed, and an alternative model is described.

Reprogramming of somatic cells to pluripotency, thereby creating induced pluripotent stem (iPS) cells, promises to transform regenerative medicine. Most instances of direct reprogramming have been achieved by forced expression of defined factors using multiple viral vectors. However, such iPS cells

Reprogramming of somatic cells to pluripotency, thereby creating induced pluripotent stem (iPS) cells, promises to transform regenerative medicine. Most instances of direct reprogramming have been achieved by forced expression of defined factors using multiple viral vectors. However, such iPS cells contain a large number of viral vector integrations, any one of which could cause unpredictable genetic dysfunction. Whereas c-Myc is dispensable for reprogramming, complete elimination of the other exogenous factors is also desired because ectopic expression of either Oct4 (also known as Pou5f1) or Klf4 can induce dysplasia. Two transient transfection-reprogramming methods have been published to address this issue. However, the efficiency of both approaches is extremely low, and neither has been applied successfully to human cells so far. Here we show that non-viral transfection of a single multiprotein expression vector, which comprises the coding sequences of c-Myc, Klf4, Oct4 and Sox2 linked with 2A peptides, can reprogram both mouse and human fibroblasts. Moreover, the transgene can be removed once reprogramming has been achieved. iPS cells produced with this non-viral vector show robust expression of pluripotency markers, indicating a reprogrammed state confirmed functionally by in vitro differentiation assays and formation of adult chimaeric mice. When the single-vector reprogramming system was combined with a piggyBac transposon, we succeeded in establishing reprogrammed human cell lines from embryonic fibroblasts with robust expression of pluripotency markers. This system minimizes genome modification in iPS cells and enables complete elimination of exogenous reprogramming factors, efficiently providing iPS cells that are applicable to regenerative medicine, drug screening and the establishment of disease models.

Transgenic expression of just four defined transcription factors (c-Myc, Klf4, Oct4 and Sox2) is sufficient to reprogram somatic cells to a pluripotent state. The resulting induced pluripotent stem (iPS) cells resemble embryonic stem cells in their properties and potential to differentiate into a sp

Transgenic expression of just four defined transcription factors (c-Myc, Klf4, Oct4 and Sox2) is sufficient to reprogram somatic cells to a pluripotent state. The resulting induced pluripotent stem (iPS) cells resemble embryonic stem cells in their properties and potential to differentiate into a spectrum of adult cell types. Current reprogramming strategies involve retroviral, lentiviral, adenoviral and plasmid transfection to deliver reprogramming factor transgenes. Although the latter two methods are transient and minimize the potential for insertion mutagenesis, they are currently limited by diminished reprogramming efficiencies. piggyBac (PB) transposition is host-factor independent, and has recently been demonstrated to be functional in various human and mouse cell lines. The PB transposon/transposase system requires only the inverted terminal repeats flanking a transgene and transient expression of the transposase enzyme to catalyse insertion or excision events. Here we demonstrate successful and efficient reprogramming of murine and human embryonic fibroblasts using doxycycline-inducible transcription factors delivered by PB transposition. Stable iPS cells thus generated express characteristic pluripotency markers and succeed in a series of rigorous differentiation assays. By taking advantage of the natural propensity of the PB system for seamless excision, we show that the individual PB insertions can be removed from established iPS cell lines, providing an invaluable tool for discovery. In addition, we have demonstrated the traceless removal of reprogramming factors joined with viral 2A sequences delivered by a single transposon from murine iPS lines. We anticipate that the unique properties of this virus-independent simplification of iPS cell production will accelerate this field further towards full exploration of the reprogramming process and future cell-based therapies.

Pluripotent cells, such as embryonic stem cells, are invaluable tools for research and can potentially serve as a source of cell- and tissue-replacement therapy. Rejection after transplantation of cells and tissue derived from embryonic stem cells is a significant obstacle to their clinical use. Rec

Pluripotent cells, such as embryonic stem cells, are invaluable tools for research and can potentially serve as a source of cell- and tissue-replacement therapy. Rejection after transplantation of cells and tissue derived from embryonic stem cells is a significant obstacle to their clinical use. Recently, human somatic cells have been reprogrammed directly to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) to yield induced pluripotent stem (iPS) cells. Human iPS cells are a potential source of patient-specific pluripotent stem cells that would bypass immune rejection. iPS cells can also be used to study diseases for which there are no adequate human in vitro or animal models. In this protocol, we describe how to establish primary human fibroblasts lines and how to derive iPS cells by retroviral transduction of reprogramming factors. Overall, it takes 2 months to complete reprogramming human primary fibroblasts starting from biopsy.

phiC31 integrase, a site-specific recombinase, can effectively mediate foreign genes bearing an attB sequence integrated into pseudo attP sites. We have previously identified two pseudo attP sites, BpsF1 and BpsM1 from the bovine genome. In this study, two new pseudo attP sites, BF4 and BF10, were d

phiC31 integrase, a site-specific recombinase, can effectively mediate foreign genes bearing an attB sequence integrated into pseudo attP sites. We have previously identified two pseudo attP sites, BpsF1 and BpsM1 from the bovine genome. In this study, two new pseudo attP sites, BF4 and BF10, were discovered using half-nested inverse PCR from cow fibroblasts. The genomic locations of these two pseudo attP sites were identified by direct sequencing and a BLAST search, and it was confirmed that they reside at positions 4q31 and 10q35 by fluorescence in situ hybridization analysis. Subsequently, the distinct integration frequencies of the four pseudo attP sites were examined. The BF4 site was identified as a hotspot where site-specific integration occurred in most of the cell clones examined, accounting for 74% (42/57) of the integration; much more than the integration frequency for BF10 (7%; 4/57), BpsF1 (7%; 4/57) and BpsM1 (0/57). Interestingly, similar to other hotspots identified in the human and mouse genomes, in which transgenes integrated at hotspots result in high expression, the GFP gene integrated at hotspot BF4 was expressed at high levels in cow fibroblasts, as confirmed by fluorescence microscopy and FACS analysis. Furthermore, ELISA showed that the expression level of the GFP gene integrated at the BF4 site averaged approximately 328 microg x mg(-1), which is more than twofold higher than that integrated at the BF10 site. This study suggests that somatic cells carrying a desired gene integrated at the BF4 site can be used as nuclear donors to generate valuable transgenic animals by nuclear transfer.

PURPOSE: Chordomas are rare, malignant bone neoplasms in which the pathogenic mechanisms remain unknown. Interestingly, tuberous sclerosis complex (TSC) is the only syndrome in which the incidence of chordomas has been described. We previously reported the pathogenic role of the TSC genes in TSC-ass

PURPOSE: Chordomas are rare, malignant bone neoplasms in which the pathogenic mechanisms remain unknown. Interestingly, tuberous sclerosis complex (TSC) is the only syndrome in which the incidence of chordomas has been described. We previously reported the pathogenic role of the TSC genes in TSC-associated chordomas. In this study, we investigated whether aberrant TSC/mammalian target of rapamycin complex 1 (mTORC1) signaling pathway is associated with sporadic chordomas. EXPERIMENTAL DESIGN: We assessed the status of mTORC1 signaling in primary tumors/cell lines of sacral chordomas and further examined upstream of mTORC1 signaling, including the PTEN (phosphatase and tensin homologue deleted on chromosome ten) tumor suppressor. We also tested the efficacy of the mTOR inhibitor rapamycin on signaling and growth of chordoma cell lines. RESULTS: Sporadic sacral chordoma tumors and cell lines examined commonly displayed hyperactivated Akt and mTORC1 signaling. Strikingly, expression of PTEN, a negative regulator of mTORC1 signaling, was not detected or significantly reduced in chordoma-derived cell lines and primary tumors. Furthermore, rapamycin inhibited mTORC1 activation and suppressed proliferation of chordoma-derived cell line. CONCLUSIONS: Our results suggest that loss of PTEN as well as other genetic alterations that result in constitutive activation of Akt/mTORC1 signaling may contribute to the development of sporadic chordomas. More importantly, a combination of Akt and mTORC1 inhibition may provide clinical benefits to chordoma patients.

Safety issues concerning the risk of malignancy formation and immune response to viral vectors were raised in initial gene therapy trials. In contrast, non-viral gene delivery methods have long been offside. We therefore explore a non-viral gene transfer approach for the treatment of hemophilia B. M

Safety issues concerning the risk of malignancy formation and immune response to viral vectors were raised in initial gene therapy trials. In contrast, non-viral gene delivery methods have long been offside. We therefore explore a non-viral gene transfer approach for the treatment of hemophilia B. METHODS: First, we constructed a strong liver-specific expression plasmid for human factor IX (FIX). Next, we tested the vector by injecting two doses under hydrodynamic conditions into the tail veins of FIX knockout mice. RESULTS: A single injection resulted in an increase in FIX expression over 100% of normal plasma levels. The FIX resulted fully functional. Further, no anti-FIX antibodies were observed and expression levels were vector dose dependent. CONCLUSION: The high expression obtained in small animals give hope for further development of non-viral gene transfer for the treatment of hemophilia B in humans.

High-resolution structural magnetic resonance imaging (MRI) has become a powerful tool in human cerebellar lesion studies. Structural MRI is helpful to analyse the localisation and extent of cerebellar lesions and to determine possible extracerebellar involvement. Functionally meaningful correlation

High-resolution structural magnetic resonance imaging (MRI) has become a powerful tool in human cerebellar lesion studies. Structural MRI is helpful to analyse the localisation and extent of cerebellar lesions and to determine possible extracerebellar involvement. Functionally meaningful correlations between a cerebellar lesion site and behavioural data can be obtained both in subjects with degenerative as well as focal cerebellar disorders. In this review, examples are presented which demonstrate that MRI-based lesion-symptom mapping is helpful to study the function of cerebellar cortex and cerebellar nuclei. Behavioural measures were used which represent two main areas of cerebellar function, that is, motor coordination and motor learning. One example are correlations with clinical data which are in good accordance with the known functional compartmentalisation of the cerebellum in three sagittal zones: In patients with cerebellar cortical degeneration ataxia of stance and gait was correlated with atrophy of the medial (and intermediate) cerebellum, oculomotor disorders with the medial, dysarthria with the intermediate and limb ataxia with atrophy of the intermediate and lateral cerebellum. Similar findings were obtained in patients with focal lesions. In addition, in patients with acute focal lesions, a somatotopy in the superior cerebellar cortex was found which is in close relationship to animal data and functional MRI data in healthy control subjects. Finally, comparison of data in patients with acute and chronic focal lesions revealed that lesion site appears to be critical for motor recovery. Recovery after lesions to the nuclei of the cerebellum was less complete. Another example which extended knowledge about functional localisation within the cerebellum is classical conditioning of the eyeblink response, a simple form of motor learning. In healthy subjects, learning rate was related to the volume of the cortex of the posterior cerebellar lobe. In patients with focal cerebellar lesions, acquisition of eyeblink conditioning was significantly reduced in lesions including the cortex of the superior posterior lobe, but not the inferior posterior lobe. Disordered timing of conditioned eyeblink responses correlated with lesions of the anterior lobe. Findings are in good agreement with the animal literature. Different parts of the cerebellar cortex may be involved in acquisition and timing of conditioned eyeblink responses in humans. These examples demonstrate that MRI-based lesion-symptom mapping is helpful to study the contribution of functionally relevant cerebellar compartments in motor control and recovery in patients with cerebellar disease. In addition, information about the function of cerebellar cortex and nuclei can be gained.

Spinal kyphosis has been speculated to participate in the increased frequency of gastroesophageal reflux disease (GERD) in patients with osteoporosis. The present study provides further evidence that increases in lumbar kyphosis and number of vertebral fractures represent very important risk factors

Spinal kyphosis has been speculated to participate in the increased frequency of gastroesophageal reflux disease (GERD) in patients with osteoporosis. The present study provides further evidence that increases in lumbar kyphosis and number of vertebral fractures represent very important risk factors for GERD in patients with osteoporosis. INTRODUCTION: Osteoporosis and spinal kyphosis have been speculated to participate in the increased frequency of gastroesophageal reflux disease (GERD). The present study examined whether GERD in patients with osteoporosis is affected by spinal factors including spinal kyphosis in the presence of oral pharmacotherapies. METHODS: Subjects comprised 112 patients with osteoporosis (mean age, 78 years) who responded to the Frequency Scale for Symptoms of GERD (FSSG) questionnaire, regardless of complaints. Relationships between total FSSG score and number of vertebral fractures, angles of kyphosis, use of bisphosphonates and nonsteroidal anti-inflammatory drugs (NSAIDs), and total number of oral medicines per day were evaluated. Logistic regression identified factors associated with GERD. RESULTS: Bisphosphonates and NSAIDs did not affect total FSSG score. Total FSSG score showed significant positive correlations with total number of medicines (r = 0.283, p = 0.0025), angle of lumbar kyphosis (r = 0.576, p = 0.0001), and numbers of thoracic vertebral fractures (r = 0.214, p = 0.0232) and lumbar vertebral fractures (r = 0.471, p < 0.0001). Angle of lumbar kyphosis and number of lumbar vertebral fractures were identified by multivariate analysis as indices affecting the presence of GERD. CONCLUSION: Increases in angle of lumbar kyphosis and number of lumbar vertebral fractures may5 represent very important risk factors for GERD in osteoporotic patients.

Background: Physical and aerobic capacity are extremely limited in dialysis patients, but it is uncertain whether or not exercise training is safe or has beneficial effects. This study aimed to assess the effects of exercise training on functional capacity and quality of life of hemodialysis patient

Background: Physical and aerobic capacity are extremely limited in dialysis patients, but it is uncertain whether or not exercise training is safe or has beneficial effects. This study aimed to assess the effects of exercise training on functional capacity and quality of life of hemodialysis patients. Methods: Ten hemodialysis patients (7 men, 3 women, aged 37 /- 7 years) free from severe comorbidities were recruited. They underwent training sessions (up to 90 minutes of submaximal exercise) twice a week, on nondialysis days, for 12 months. At baseline and after the physical training program, all patients underwent biochemistry, cardiopulmonary exercise test, echocardiography and a self-rated health test (SF-36). Results: At baseline, dialysis patients showed impaired VO2 uptake (20.6 /- 5.0 ml/kg/min vs. 34.2 /- 6.0 ml/kg/min, p<0.001) and peak working capacity (115 /- 36 W vs. 192 /- 46.7 W, p<0.001) compared with normal controls. Following the training program, both peak VO2 (20.4 /- 4.9 ml/kg/min vs. 25.1 /- 6.5 ml/kg/min, p<0.05), VO2 at anaerobic threshold (12.8 /- 1.9 ml/kg/min vs. 15.1 /- 3.8 ml/kg/min, p<0.05), peak working capacity (113 /- 33 W vs. 134 /- 37 W, p<0.01) and SF-36 scores improved. No side effects related to intervention occurred. Conclusions: Dialysis patients showed impaired muscular exercise capacity, but 12 months of moderate exercise training was able to improve their physical function, aerobic capacity and quality of life. Our study suggests that mild, regular physical activity should be recommended and encouraged as an important aspect of the care of selected dialysis patients.

Background: Extracellular matrix accumulation, epithelial-to-mesenchymal transition, tubular atrophy and loss of peritubular capillary network are hallmarks of tubulointerstitial injury in progressive renal diseases. In this study, we analyzed endostatin expression in kidneys subjected to unilateral

Background: Extracellular matrix accumulation, epithelial-to-mesenchymal transition, tubular atrophy and loss of peritubular capillary network are hallmarks of tubulointerstitial injury in progressive renal diseases. In this study, we analyzed endostatin expression in kidneys subjected to unilateral ureteral obstruction (UUO). Methods: Collagen XVIII mRNA expression was evaluated by real-time polymerase chain reaction (PCR). Endostatin and CD31 protein levels were analyzed by Western blot and immunohistochemistry. In vitro quantification of collagen XVIII and fibrosis-related genes in HK2 cells was performed by real-time PCR. Results: UUO significantly increased collagen XVIII mRNA expression and released a 30-kDa endostatin fragment. Immunohistochemistry revealed endostatin expression increased in injured tissue, mainly on tubular cells. Of interest, expression of CD31 was significantly reduced by UUO. Endostatin administration in vitro did not modify the expression of genes related to fibrosis development. However, in vitro TGF-beta1 administration induced expression of collagen XVIII/endostatin mRNA in human tubular cells. Conclusion: Endostatin is expressed during the progression of renal fibrosis in vitro and in vivo, suggesting a role for endostatin in development of tubulointerstitial injury.

An experimental test facility is developed for use by stroke patients in order to improve sensory-motor function of their upper limb. Subjects are seated at the workstation and their task is to repeatedly follow reaching trajectories that are projected onto a target above their arm. To do this they

An experimental test facility is developed for use by stroke patients in order to improve sensory-motor function of their upper limb. Subjects are seated at the workstation and their task is to repeatedly follow reaching trajectories that are projected onto a target above their arm. To do this they use voluntary control with the addition of electrical stimulation mediated by advanced control schemes applied to muscles in their impaired shoulder and arm. Full details of the design of the workstation and its periphery systems are given, together with a description of its use during the treatment of stroke patients.

In Europe there are about 300,000 paraplegics and in every country approximately 1000 new cases per year. Treatment requires a multidisciplinary approach with scientific cooperation targeted to exchange personal knowledge and expertise. At present a completely disrupted spinal cord cannot heal for r

In Europe there are about 300,000 paraplegics and in every country approximately 1000 new cases per year. Treatment requires a multidisciplinary approach with scientific cooperation targeted to exchange personal knowledge and expertise. At present a completely disrupted spinal cord cannot heal for recovery of motor and/or sensory functioning, although some promising treatment modalities in laboratory animal experiments have been reported. No interventional stem cell procedure so far has shown evidence to restore impaired functioning in human paraplegics. However, functional electrical stimulation (FES) via an implanted neuroprosthesis (SUAW concept) and central nervous system-peripheral nervous system (CNS-PNS) connection have successfully been used for alternative compensatory strategies for voluntary locomotion. This report is to analyse the authors' experience from two European projects in paraplegic. Factors will be identified that might have caused the one or other pitfall since so far both surgical reconstructive procedures have not been adopted by rehabilitation physicians and/or restorative (neuro-)surgeons despite the promising functional results we have achieved. Unexpected plasticity of single neurons following CNS-PNS by-pass procedures is discussed. Future interventions, for example the present phase 1 prospective multiple centre study on the side effects, effectiveness, and reliability of intrathecal treatment of anti-Nogo-A antibodies, are presented and the Chinese stem cell implantation is critically reviewed.