The endogenous hydrogen sulfide producing enzyme cystathionine-β synthase contributes to visceral hypersensitivity in a rat model of irritable bowel syndrome

<p>Abstract</p> <p>Background</p> <p>The pathogenesis of visceral hypersensitivity, a characteristic pathophysiological feature of irritable bowel syndrome (IBS), remains elusive. Recent studies suggest a role for hydrogen sulfide (H₂S) in pain signaling but this has not been well studied in visceral models of hyperalgesia. We therefore determined the role for the endogenous H₂S producing enzyme cystathionine-β-synthetase (CBS) in a validated rat model of IBS-like chronic visceral hyperalgesia (CVH). CVH was induced by colonic injection of 0.5% acetic acid (AA) in 10-day-old rats and experiments were performed at 8–10 weeks of age. Dorsal root ganglion (DRG) neurons innervating the colon were labeled by injection of DiI (1,1'-dioleyl-3,3,3',3-tetramethylindocarbocyanine methanesulfonate) into the colon wall.</p> <p>Results</p> <p>In rat DRG, CBS-immunoreactivity was observed in approximately 85% of predominantly small- and medium-sized neurons. Colon specific DRG neurons revealed by retrograde labeling DiI were all CBS-positive. CBS-positive colon neurons co-expressed TRPV1 or P2X3 receptors. Western blotting analysis showed that CBS expression was significantly increased in colon DRGs 8 weeks after neonatal AA-treatment. Furthermore, the CBS inhibitor hydroxylamine markedly attenuated the abdominal withdrawal reflex scores in response to colorectal distention in rats with CVH. By contrast, the H₂S donor NaHS significantly enhanced the frequency of action potentials of colon specific DRG neurons evoked by 2 times rheobase electrical stimulation.</p> <p>Conclusion</p> <p>Our results suggest that upregulation of CBS expression in colonic DRG neurons and H₂S signaling may play an important role in developing CVH, thus identifying a specific neurobiological target for the treatment of CVH in functional bowel syndromes.</p

DNA Immunization with Fusion of CTLA-4 to Hepatitis B Virus (HBV) Core Protein Enhanced Th2 Type Responses and Cleared HBV with an Accelerated Kinetic

BACKGROUND: Typically, DNA immunization via the intramuscular route induces specific, Th1-dominant immune responses. However, plasmids expressing viral proteins fused to cytotoxic T lymphocyte antigen 4 (CTLA-4) primed Th2-biased responses and were able to induced effective protection against viral challenge in the woodchuck model. Thus, we addressed the question in the mouse model how the Th1/Th2 bias of primed immune responses by a DNA vaccine influences hepatitis B virus (HBV) clearance. PRINCIPAL FINDINGS: Plasmids expressing HBV core protein (HBcAg) or HBV e antigen and HBcAg fused to the extracellular domain of CTLA-4 (pCTLA-4-HBc), CD27, and full length CD40L were constructed. Immunizations of these DNA plasmids induced HBcAg-specific antibody and cytotoxic T-cell responses in mice, but with different characteristics regarding the titers and subtypes of specific antibodies and intensity of T-cell responses. The plasmid pHBc expressing HBcAg induced an IgG2a-dominant response while immunizations of pCTLA-4-HBc induced a balanced IgG1/IgG2a response. To assess the protective values of the immune responses of different characteristics, mice were pre-immunized with pCTLA-4-HBc and pHBc, and challenged by hydrodynamic injection (HI) of pAAV/HBV1.2. HBV surface antigen (HBsAg) and DNA in peripheral blood and HBcAg in liver tissue were cleared with significantly accelerated kinetics in both groups. The clearance of HBsAg was completed within 16 days in immunized mice while more than 50% of the control mice are still positive for HBsAg on day 22. Stronger HBcAg-specific T-cell responses were primed by pHBc correlating with a more rapid decline of HBcAg expression in liver tissue, while anti-HBs antibody response developed rapidly in the mice immunized with pCTLA-4-HBc, indicating that the Th1/Th2 bias of vaccine-primed immune responses influences the mode of viral clearance. CONCLUSION: Viral clearance could be efficiently achieved by Th1/Th2-balanced immune response, with a small but significant shift in T-cell and B-cell immune responses

Hypoxia and hypoglycaemia in Ewing's sarcoma and osteosarcoma: regulation and phenotypic effects of Hypoxia-Inducible Factor

<p>Abstract</p> <p>Background</p> <p>Hypoxia regulates gene expression via the transcription factor HIF (Hypoxia-Inducible Factor). Little is known regarding HIF expression and function in primary bone sarcomas. We describe HIF expression and phenotypic effects of hypoxia, hypoglycaemia and HIF in Ewing's sarcoma and osteosarcoma.</p> <p>Methods</p> <p>HIF-1α and HIF-2α immunohistochemistry was performed on a Ewing's tumour tissue array. Ewing's sarcoma and osteosarcoma cell lines were assessed for HIF pathway induction by Western blot, luciferase assay and ELISA. Effects of hypoxia, hypoglycaemia and isoform-specific HIF siRNA were assessed on proliferation, apoptosis and migration.</p> <p>Results</p> <p>17/56 Ewing's tumours were HIF-1α-positive, 15 HIF-2α-positive and 10 positive for HIF-1α and HIF-2α. Expression of HIF-1α and cleaved caspase 3 localised to necrotic areas. Hypoxia induced HIF-1α and HIF-2α in Ewing's and osteosarcoma cell lines while hypoglycaemia specifically induced HIF-2α in Ewing's. Downstream transcription was HIF-1α-dependent in Ewing's sarcoma, but regulated by both isoforms in osteosarcoma. In both cell types hypoglycaemia reduced cellular proliferation by ≥ 45%, hypoxia increased apoptosis and HIF siRNA modulated hypoxic proliferation and migration.</p> <p>Conclusions</p> <p>Co-localisation of HIF-1α and necrosis in Ewing's sarcoma suggests a role for hypoxia and/or hypoglycaemia in <it>in vivo </it>induction of HIF. <it>In vitro </it>data implicates hypoxia as the primary HIF stimulus in both Ewing's and osteosarcoma, driving effects on proliferation and apoptosis. These results provide a foundation from which to advance understanding of HIF function in the pathobiology of primary bone sarcomas.</p

Significance of Input Correlations in Striatal Function

The striatum is the main input station of the basal ganglia and is strongly associated with motor and cognitive functions. Anatomical evidence suggests that individual striatal neurons are unlikely to share their inputs from the cortex. Using a biologically realistic large-scale network model of striatum and cortico-striatal projections, we provide a functional interpretation of the special anatomical structure of these projections. Specifically, we show that weak pairwise correlation within the pool of inputs to individual striatal neurons enhances the saliency of signal representation in the striatum. By contrast, correlations among the input pools of different striatal neurons render the signal representation less distinct from background activity. We suggest that for the network architecture of the striatum, there is a preferred cortico-striatal input configuration for optimal signal representation. It is further enhanced by the low-rate asynchronous background activity in striatum, supported by the balance between feedforward and feedback inhibitions in the striatal network. Thus, an appropriate combination of rates and correlations in the striatal input sets the stage for action selection presumably implemented in the basal ganglia

Two-Photon Microscopy for Non-Invasive, Quantitative Monitoring of Stem Cell Differentiation

BACKGROUND: The engineering of functional tissues is a complex multi-stage process, the success of which depends on the careful control of culture conditions and ultimately tissue maturation. To enable the efficient optimization of tissue development protocols, techniques suitable for monitoring the effects of added stimuli and induced tissue changes are needed. METHODOLOGY/PRINCIPAL FINDINGS: Here, we present the quantitative use of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) as a noninvasive means to monitor the differentiation of human mesenchymal stem cells (hMSCs) using entirely endogenous sources of contrast. We demonstrate that the individual fluorescence contribution from the intrinsic cellular fluorophores NAD(P)H, flavoproteins and lipofuscin can be extracted from TPEF images and monitored dynamically from the same cell population over time. Using the redox ratio, calculated from the contributions of NAD(P)H and flavoproteins, we identify distinct patterns in the evolution of the metabolic activity of hMSCs maintained in either propagation, osteogenic or adipogenic differentiation media. The differentiation of these cells is mirrored by changes in cell morphology apparent in high resolution TPEF images and by the detection of collagen production via SHG imaging. Finally, we find dramatic increases in lipofuscin levels in hMSCs maintained at 20% oxygen vs. those in 5% oxygen, establishing the use of this chromophore as a potential biomarker for oxidative stress. CONCLUSIONS/SIGNIFICANCE: In this study we demonstrate that it is possible to monitor the metabolic activity, morphology, ECM production and oxidative stress of hMSCs in a non-invasive manner. This is accomplished using generally available multiphoton microscopy equipment and simple data analysis techniques, such that the method can widely adopted by laboratories with a diversity of comparable equipment. This method therefore represents a powerful tool, which enables researchers to monitor engineered tissues and optimize culture conditions in a near real time manner

An Antagomir to MicroRNA Let7f Promotes Neuroprotection in an Ischemic Stroke Model

We previously showed that middle-aged female rats sustain a larger infarct following experimental stroke as compared to younger female rats, and paradoxically, estrogen treatment to the older group is neurotoxic. Plasma and brain insulin-like growth factor-1 (IGF-1) levels decrease with age. However, IGF-1 infusion following stroke, prevents estrogen neurotoxicity in middle-aged female rats. IGF1 is neuroprotective and well tolerated, but also has potentially undesirable side effects. We hypothesized that microRNAs (miRNAs) that target the IGF-1 signaling family for translation repression could be alternatively suppressed to promote IGF-1-like neuroprotection. Here, we report that two conserved IGF pathway regulatory microRNAs, Let7f and miR1, can be inhibited to mimic and even extend the neuroprotection afforded by IGF-1. Anti-mir1 treatment, as late as 4 hours following ischemia, significantly reduced cortical infarct volume in adult female rats, while anti-Let7 robustly reduced both cortical and striatal infarcts, and preserved sensorimotor function and interhemispheric neural integration. No neuroprotection was observed in animals treated with a brain specific miRNA unrelated to IGF-1 (anti-miR124). Remarkably, anti-Let7f was only effective in intact females but not males or ovariectomized females indicating that the gonadal steroid environment critically modifies miRNA action. Let7f is preferentially expressed in microglia in the ischemic hemisphere and confirmed in ex vivo cultures of microglia obtained from the cortex. While IGF-1 was undetectable in microglia harvested from the non-ischemic hemisphere, IGF-1 was expressed by microglia obtained from the ischemic cortex and was further elevated by anti-Let7f treatment. Collectively these data support a novel miRNA-based therapeutic strategy for neuroprotection following stroke

Activity-Based Funding of Hospitals and Its Impact on Mortality, Readmission, Discharge Destination, Severity of Illness, and Volume of Care: A Systematic Review and Meta-Analysis

Background: Activity-based funding (ABF) of hospitals is a policy intervention intended to re-shape incentives across health systems through the use of diagnosis-related groups. Many countries are adopting or actively promoting ABF. We assessed the effect of ABF on key measures potentially affecting patients and health care systems: mortality (acute and post-acute care); readmission rates; discharge rate to post-acute care following hospitalization; severity of illness; volume of care. &nbsp; &nbsp; Methods: We undertook a systematic review and meta-analysis of the worldwide evidence produced since 1980. We included all studies reporting original quantitative data comparing the impact of ABF versus alternative funding systems in acute care settings, regardless of language. We searched 9 electronic databases (OVID MEDLINE, EMBASE, OVID Healthstar, CINAHL, Cochrane CENTRAL, Health Technology Assessment, NHS Economic Evaluation Database, Cochrane Database of Systematic Reviews, and Business Source), hand-searched reference lists, and consulted with experts. Paired reviewers independently screened for eligibility, abstracted data, and assessed study credibility according to a pre-defined scoring system, resolving conflicts by discussion or adjudication. &nbsp; &nbsp; Results: Of 16,565 unique citations, 50 US studies and 15 studies from 9 other countries proved eligible (i.e. Australia, Austria, England, Germany, Israel, Italy, Scotland, Sweden, Switzerland). We found consistent and robust differences between ABF and no-ABF in discharge to post-acute care, showing a 24% increase with ABF (pooled relative risk = 1.24, 95% CI 1.18–1.31). Results also suggested a possible increase in readmission with ABF, and an apparent increase in severity of illness, perhaps reflecting differences in diagnostic coding. Although we found no consistent, systematic differences in mortality rates and volume of care, results varied widely across studies, some suggesting appreciable benefits from ABF, and others suggesting deleterious consequences. &nbsp; &nbsp; Conclusions: Transitioning to ABF is associated with important policy- and clinically-relevant changes. Evidence suggests substantial increases in admissions to post-acute care following hospitalization, with implications for system capacity and equitable access to care. High variability in results of other outcomes leaves the impact in particular settings uncertain, and may not allow a jurisdiction to predict if ABF would be harmless. Decision-makers considering ABF should plan for likely increases in post-acute care admissions, and be aware of the large uncertainty around impacts on other critical outcomes

Characterizing Acupuncture Stimuli Using Brain Imaging with fMRI - A Systematic Review and Meta-Analysis of the Literature

Background The mechanisms of action underlying acupuncture, including acupuncture point specificity, are not well understood. In the previous decade, an increasing number of studies have applied fMRI to investigate brain response to acupuncture stimulation. Our aim was to provide a systematic overview of acupuncture fMRI research considering the following aspects: 1) differences between verum and sham acupuncture, 2) differences due to various methods of acupuncture manipulation, 3) differences between patients and healthy volunteers, 4) differences between different acupuncture points. Methodology/Principal Findings We systematically searched English, Chinese, Korean and Japanese databases for literature published from the earliest available up until September 2009, without any language restrictions. We included all studies using fMRI to investigate the effect of acupuncture on the human brain (at least one group that received needle-based acupuncture). 779 papers were identified, 149 met the inclusion criteria for the descriptive analysis, and 34 were eligible for the meta-analyses. From a descriptive perspective, multiple studies reported that acupuncture modulates activity within specific brain areas, including somatosensory cortices, limbic system, basal ganglia, brain stem, and cerebellum. Meta-analyses for verum acupuncture stimuli confirmed brain activity within many of the regions mentioned above. Differences between verum and sham acupuncture were noted in brain response in middle cingulate, while some heterogeneity was noted for other regions depending on how such meta-analyses were performed, such as sensorimotor cortices, limbic regions, and cerebellum. Conclusions Brain response to acupuncture stimuli encompasses a broad network of regions consistent with not just somatosensory, but also affective and cognitive processing. While the results were heterogeneous, from a descriptive perspective most studies suggest that acupuncture can modulate the activity within specific brain areas, and the evidence based on meta-analyses confirmed some of these results. More high quality studies with more transparent methodology are needed to improve the consistency amongst different studies