Randomized Controlled Trial of Physical Exercise in Diabetic Veterans With Length-Dependent Distal Symmetric Polyneuropathy

Rationale: Physical exercise is an essential adjunct to the management of patients with type 2 diabetes mellitus. Therapeutic interventions that improve blood flow to peripheral nerves, such as exercise, may slow the progression of neuropathy in the diabetic patient.Aims: This randomized clinical trial was conducted to determine whether a structured program of aerobic, isokinetic strength, or the combination of aerobic–isokinetic strength exercise intervention alters peripheral nerve function in glycemic-controlled diabetic patients with advanced length-dependent distal symmetric polyneuropathy.Methods: Forty-five patients with type 2 diabetes mellitus exhibiting tight glycemic control (HbA1c intergroup range 7.2–8.0%) were randomized by block design across four experimental groups: sedentary controls (n = 12), aerobic exercise (n = 11), isokinetic strength (n = 11), or the combination of aerobic–isokinetic strength training (n = 11). Patients randomized to training groups exercised 3× per week for 12 weeks, whereas patients randomized to the sedentary control group received standard of care. To minimize attention and educational bias, all patients attended a 12-session health promotion educational series. At baseline, immediately following intervention, and again at 12-week post-intervention, detailed nerve conduction studies were conducted as a primary outcome measure. At these same intervals, all patients completed as secondary measures quantitative sensory testing, symptom-limited treadmill stress tests, and a Short-Form 36-Veterans Questionnaire (SF-36V).Results: Of the 45 patients randomized into this study, 37 (82%) had absent sural nerve responses, 19 (42%) had absent median sensory nerve responses, and 17 (38%) had absent ulnar sensory nerve responses. By comparison, responses from tibial nerves were absent in only three (7%) subjects while responses from peroneal nerves were absent in five (11%) subjects. Eleven (92%) of 12 patients that had volunteered to be biopsied exhibited abnormal levels of epidermal nerve fiber densities. Exercise, regardless of type, did not alter sensory or motor nerve electrodiagnostic findings among those patients exhibiting measurable responses (ANOVA). There was, however, a modest (p = 0.01) beneficial effect of exercise on sensory nerve function (Fisher’s Exact Test). Importantly, the beneficial effect of exercise on sensory nerve function was enhanced (p = 0.03) during the post-intervention interval. In addition, three of six patients that had undergone exercise intervention exhibited a marked 1.9 ± 0.3-fold improvement in epidermal nerve fiber density. By comparison, none of three sedentary patients whom agreed to be biopsied a second time showed improvement in epidermal nerve fiber density. Compared to baseline values within groups, and compared with sedentary values across groups, neither aerobic, isokinetic strength, or the combination of aerobic–isokinetic strength exercise intervention altered peak oxygen uptake. Patients that underwent aerobic or the combined aerobic–isokinetic strength exercise intervention, however, demonstrated an increase in treadmill test duration that was sustained over the 12-week post-intervention period.Conclusion: A 12-week course of physical exercise, regardless of type, does not alter sensory or motor nerve electrodiagnostic findings. In a subset of patients, a short-term structured program of aerobic exercise may selectively improve sensory nerve fiber function. Large-scale exercise lifestyle intervention trials are warranted to further evaluate the impact of aerobic exercise on sensory nerve fiber function in diabetic neuropathic patients.Clinical Trial Registration:www.ClinicalTrials.gov, identifier NCT00955201

Transforming Growth Factor-b2 Induces Synthesis and Secretion of Endothelin-1 in Human Trabecular Meshwork Cells

PURPOSE. Analysis of aqueous humor from patients with primary open-angle glaucoma (POAG) revealed marked increases in the content of endothelin-1 (ET-1) and transforming growth factor-beta (TGF-b). We determined the consequences of TGF-b signaling on ET-1 expression and secretion by human trabecular meshwork (TM) cells. METHODS. Primary or transformed (NTM5 and GTM3) human TM cells conditioned in serum-free media were incubated in the absence or presence of TGF-b1 or -b2. Relative changes in preproendothelin (ppET)-1 mRNA content and secreted ET-1 peptide were quantified by real-time PCR and ELISA, respectively. In some experiments, TGF-b or ET-1 receptor antagonists, or Rho G-protein inhibitors, were evaluated for effects on TGF-b signaling. Filamentous actin organization was visualized by phalloidin. RESULTS. Primary or transformed human TM cells cultured in the presence of TGF-b1 or -b2 exhibit a marked (>8-fold) increase in ppET-1 mRNA content compared to vehicle controls. Coincubation with SB-505124, an inhibitor of TGFbRI/ALK-5 signaling, prevented TGF-b-mediated ppET-1 mRNA expression. In contrast, coincubation with ET A or ET B (BQ-788) receptor antagonists had no effect on TGF-b-mediated ppET-1 mRNA expression. TGF-b1 and -b2 each elicited a robust (>7-fold) secretion of ET-1 while enhancing stress fiber organization. Inhibition of Rho signaling attenuated TGF-b-mediated increases in ppET-1 mRNA content, ET-1 secretion, and stress fiber organization. CONCLUSIONS. TGF-b, signaling through the TGFbRI/ALK-5 receptor, elicits marked increases in ET-1 mRNA content and ET-1 secretion from cultured primary or transformed human TM cells. Elevated levels of TGF-b2 present in AH of POAG patients may elevate intraocular pressure, in part, by eliciting aberrant Rho G-protein dependent cell contraction, and increasing ET-1 synthesis and secretion, in human TM cells. (Invest Ophthalmol Vis Sci. 2012;53:5279-5286) DOI:10.1167/iovs.11-9289 P rimary open-angle glaucoma (POAG) is one of the most common causes of blindness worldwide, affecting over 2 million individuals 45 years or older in the United States. 1 In POAG patients, irreversible loss of peripheral vision frequently is associated with a pathologic elevation of intraocular pressure (IOP). 2 Clinically, elevated IOP remains a poorly understood hallmark of POAG. In healthy eyes, normal IOP is maintained through a balance between production and outflow of aqueous humor (AH). In adults, the majority (>50%) of AH exits the eye by a conventional outflow pathway involving the trabecular meshwork (TM) at the iridocorneal angle. 3 TM cells regulate AH outflow facility partly through contraction and relaxation of their actin cytoskeleton. Under pathologic conditions, chronic aberrant contraction of TM cells increases resistance to AH outflow, leading to abnormal and sustained elevation of IOP. The mechanism that promotes harmful chronic aberrant TM cell contraction in POAG remains unknown, but may involve dysregulation of small monomeric Rho G-protein mediated organization of the actin cytoskeleton. 23-26 Levels of transforming growth factor (TGF)-b2, a cytokine known to promote synthesis and release of ECM components, similarly are increased aberrantly in AH from POAG patients. 41 Emerging evidence strongly supports a pathologic association between either ET-1 or TGF-b2 with elevated IOP in the 5279 Downloaded from iovs.arvojournals.org on 06/29/2019 pathogenesis of POAG. Mechanisms responsible for regulating endogenous synthesis and secretion of ET-1 and TGF-b2 within the eye currently remain unknown. A role for TGF-b in promoting transcription and release of ET-1 has been suggested. MATERIALS AND METHODS Human Trabecular Meshwork Cell Culture The use of human cadaver material in our study was approved by the Edward Hines Jr. VA and Loyola University Chicago institutional review boards in compliance with the tenets of the Declaration of Helsinki. Fresh cadaver corneoscleral rims were obtained (Illinois Eye Bank, Chicago, IL) at the time of corneal transplant and primary human TM cells were prepared using a collagenase-free procedure as described previously. Treatment Subconfluent primary or transformed human TM cells were cultured for 24 hours in serum-free media before treatment. Recombinant human TGF-b1 or TGF-b2 (Cell Signaling Technology, Danvers, MA) was reconstituted as a stock solution in 4 mM HCl containing 0.1% BSA for 30 minutes at 238C before use. Serum-starved cultures were treated (24 hours) in the absence (vehicle, 200 nM HCl) or presence (5 ng/mL) of TGF-b1 or TGF-b2 in fresh serum-free media. TM cell viability was routinely determined by Trypan Blue dye exclusion and was consistently >90%. To determine mechanism of action, GTM3 cells were co-treated (24 hours) with TGF-b2 (5 ng/mL) in the absence or presence of SB-505124 (1 lM), a TGF-b type I receptor (TGFbRI)/ activin receptor-like kinase 5 (ALK-5) inhibitor; BQ-123 (1 lM), an ET A antagonist; or BQ-788 (1 lM), an ET B antagonist. To determine the role of Rho G-proteins, TM cells were pretreated (1 hour) with chemicallyactivated lovastatin (10 lM) 3,48,50 ; GGTI-298 (10 lM, a geranylgeranyl transferase-I inhibitor) or with cell-permeable exoenzyme C3 transferase (10 lg /mL, a selective Rho G-protein subfamily inhibitor; Cytoskeleton, Denver, CO). Unless otherwise indicated, all reagents were purchased from Sigma-Aldrich. Real Time RT-PCR Total RNA was extracted from primary or transformed human TM cells using TRIzol reagent, and 5 lg were reverse-transcribed using Super Script III First Strand Synthesis system (Life Technologies) as described previously. Endothelin-1 ELISA The content of ET-1 in cell culture media was assessed using a commercially-available ELISA kit (R&D Systems, Minneapolis, MN) according to manufacturer's instructions. The working range for this human-specific ET-1 ELISA kit is 0.39-25 pg/ml. Media from primary or transformed human TM cells cultured in 12-well cell culture plates were harvested, centrifuged (700g 3 5 minutes) to remove particulate, and aliquots (75 lL) were added to microtiter wells precoated with a monoclonal antibody against human ET-1. Samples were read at 450 nm with a 540 nm correction, and results expressed as pg of ET-1. Filamentous Actin Staining NTM5 or GTM3 cells were cultured on Nunc Lab-Tek II chambered slides overnight (24 hours) in serum-free DMEM and treated 3 24 hours without (vehicle, 200 nM HCl) or with 5 ng/mL TGF-b1 or TGF-b2. Some cultures were pretreated (1 hour) with chemically-activated lovastatin (10 lM) or GGTI-298 (10 lM) before TGF-b2 treatment. Treated cells were fixed 315 minutes at 238C by immersion in phosphate buffered (pH 7.4) 4% paraformaldehyde. Filamentous actin stress fiber organization was visualized using AlexaFluor488-conjugated phalloidin. Stained slides were mounted using Fluoroshield containing DAPI (Sigma), and visualized by confocal microscopy. Statistical Analysis Results are expressed as mean 6 SD of duplicate (primary) or triplicate (transformed) cultures, repeated at least one additional time unless otherwise specified. Parametric data were analyzed by Student's t-test or by one-way ANOVA followed by either a Dunnett's or Bonferroni's multiple comparison post-hoc analysis, as indicated. In all cases, P < 0.05 was considered statistically significant. RESULTS TGF-b2 Increases ppET-1 mRNA Content Transformed human TM cells (NTM5) conditioned overnight in serum-free media and incubated subsequently for 24 hours in the presence of TGF-b2 (5 ng/mL) exhibited a >8-fold increase in ppET-1 mRNA content compared to vehicle-treated controls The effect of TGF-b2 on relative changes in ppET-1 mRNA content was dose-and time-dependent 39,51-53 GTM3 cells also responded to 5 ng/mL TGF-b2 stimulation in a time-dependent 5280 Von Zee et al. IOVS, August 2012, Vol. 53, No. 9 Downloaded from iovs.arvojournals.org on 06/29/2019 manner, exhibiting significant and sustained changes in ppET-1 mRNA content at 6-12 hours of incubation 44 Pretreating TM cells with SIS3, a specific inhibitor of the canonical TGF-b effector Smad3, however, did not reduce TGFb2 enhanced ppET-1 mRNA expression (data not shown), suggesting the involvement a non-canonical TGF-b signaling pathway. Previously, a role for small monomeric G-proteins in the regulation of ppET-1 expression has been suggested. One mechanism by which statins indirectly inhibit small Gprotein signaling in TM cells is by limiting the availability of isoprenoid intermediates required for post-translational modification and activation of Rho G-proteins

Th17 Cell Response in SOD1G93A Mice following Motor Nerve Injury

An increased risk of ALS has been reported for veterans, varsity athletes, and professional football players. The mechanism underlying the increased risk in these populations has not been identified; however, it has been proposed that motor nerve injury may trigger immune responses which, in turn, can accelerate the progression of ALS. Accumulating evidence indicates that abnormal immune reactions and inflammation are involved in the pathogenesis of ALS, but the specific immune cells involved have not been clearly defined. To understand how nerve injury and immune responses may contribute to ALS development, we investigated responses of CD4(+) T cell after facial motor nerve axotomy (FNA) at a presymptomatic stage in a transgenic mouse model of ALS (B6SJL SOD1(G93A)). SOD1(G93A) mice, compared with WT mice, displayed an increase in the basal activation state of CD4(+) T cells and higher frequency of Th17 cells, which were further enhanced by FNA. In conclusion, SOD1(G93A) mice exhibit abnormal CD4(+) T cell activation with increased levels of Th17 cells prior to the onset of neurological symptoms. Motor nerve injury exacerbates Th17 cell responses and may contribute to the development of ALS, especially in those who carry genetic susceptibility to this disease

LSST Science Book, Version 2.0

A survey that can cover the sky in optical bands over wide fields to faint magnitudes with a fast cadence will enable many of the exciting science opportunities of the next decade. The Large Synoptic Survey Telescope (LSST) will have an effective aperture of 6.7 meters and an imaging camera with field of view of 9.6 deg^2, and will be devoted to a ten-year imaging survey over 20,000 deg^2 south of +15 deg. Each pointing will be imaged 2000 times with fifteen second exposures in six broad bands from 0.35 to 1.1 microns, to a total point-source depth of r~27.5. The LSST Science Book describes the basic parameters of the LSST hardware, software, and observing plans. The book discusses educational and outreach opportunities, then goes on to describe a broad range of science that LSST will revolutionize: mapping the inner and outer Solar System, stellar populations in the Milky Way and nearby galaxies, the structure of the Milky Way disk and halo and other objects in the Local Volume, transient and variable objects both at low and high redshift, and the properties of normal and active galaxies at low and high redshift. It then turns to far-field cosmological topics, exploring properties of supernovae to z~1, strong and weak lensing, the large-scale distribution of galaxies and baryon oscillations, and how these different probes may be combined to constrain cosmological models and the physics of dark energy.Comment: 596 pages. Also available at full resolution at http://www.lsst.org/lsst/sciboo

Erratum: Corrigendum: Sequence and comparative analysis of the chicken genome provide unique perspectives on vertebrate evolution

International Chicken Genome Sequencing Consortium. The Original Article was published on 09 December 2004. Nature432, 695–716 (2004). In Table 5 of this Article, the last four values listed in the ‘Copy number’ column were incorrect. These should be: LTR elements, 30,000; DNA transposons, 20,000; simple repeats, 140,000; and satellites, 4,000. These errors do not affect any of the conclusions in our paper. Additional information. The online version of the original article can be found at 10.1038/nature0315

Cytidine-Diphosphate Diacylglycerol Labeling as an Index of Inositol Lipid-Mediated Signal Transduction in Brain and Neural Cells

A method for assessing stimulated phosphoinositide turnover by measurement of the liponucleotide CDP-diacylglycerol is presented. The phosphoinositide signal transduction pathway consists of a sequence of reactions in which the second messengers Inositol 1,4,5-triphosphate and diacylglycerol are recycled back to phosphatidylinositol (PtdIns), which then serves to replenish the initial hydrolyzed substrate, phosphatidylinositol 4,5-bis-phosphate. Receptor-stimulated inositol lipid turnover is most commonly assessed by measurement of the accumulation of [3H]inositol-labeled inositol phosphates in the presence of Li+. The latter blocks Inositol monophosphatase and thus can lead to a depletion of intracellular inositol. Because inositol is required for resynthesis of PtdIns, the immediate precursor of PtdIns, CDP-diacylglycerol, also accumulates in the presence of agonist and Li+. Measurement of radiolabeling of this liponucleotide following Incorporation of [3H]cytidine thus forms the basis for an alternative assay for Inositol lipid turnover. The general applicability of this method may be limited, since, In brain slices, not all receptors exhibit CDP-diacylglycerol responses that are consistent with their inositol phosphate responses. In addition, in cultured neural cells, growth in inositol-free, chemically defined medium is required to maximize the Li+ -dependent CDP-diacylglycerol response. A major advantage of this method may be its ability to provide insight Into the regulation of phosphoinositide turnover since this method uniquely reflects slowing of the regenerative cycle. Such in vitro studies may have relevance to the in vivo action of Li+ as a psychotherapeutic agent.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30531/1/0000163.pd

Forced Exercise Preconditioning Attenuates Experimental Autoimmune Neuritis by Altering T1 Lymphocyte Composition and Egress

A short-term exposure to moderately intense physical exercise affords a novel measure of protection against autoimmune-mediated peripheral nerve injury. Here, we investigated the mechanism by which forced exercise attenuates the development and progression of experimental autoimmune neuritis (EAN), an established animal model of Guillain–Barré syndrome. Adult male Lewis rats remained sedentary (control) or were preconditioned with forced exercise (1.2 km/day × 3 weeks) prior to P2-antigen induction of EAN. Sedentary rats developed a monophasic course of EAN beginning on postimmunization day 12.3 ± 0.2 and reaching peak severity on day 17.0 ± 0.3 ( N  = 12). By comparison, forced-exercise preconditioned rats exhibited a similar monophasic course but with significant ( p  < .05) reduction of disease severity. Analysis of popliteal lymph nodes revealed a protective effect of exercise preconditioning on leukocyte composition and egress. Compared with sedentary controls, forced exercise preconditioning promoted a sustained twofold retention of P2-antigen responsive leukocytes. The percentage distribution of pro-inflammatory (T h 1) lymphocytes retained in the nodes from sedentary EAN rats (5.1 ± 0.9%) was significantly greater than that present in nodes from forced-exercise preconditioned EAN rats (2.9 ± 0.6%) or from adjuvant controls (2.0 ± 0.3%). In contrast, the percentage of anti-inflammatory (T h 2) lymphocytes (7–10%) and that of cytotoxic T lymphocytes (∼20%) remained unaltered by forced exercise preconditioning. These data do not support an exercise-inducible shift in T h 1:T h 2 cell bias. Rather, preconditioning with forced exercise elicits a sustained attenuation of EAN severity, in part, by altering the composition and egress of autoreactive proinflammatory (T h 1) lymphocytes from draining lymph nodes