Glycoprotein Ib activation by thrombin stimulates the energy metabolism in human platelets

<div><p>Thrombin-induced platelet activation requires substantial amounts of ATP. However, the specific contribution of each ATP-generating pathway <i>i</i>.<i>e</i>., oxidative phosphorylation (OxPhos) versus glycolysis and the biochemical mechanisms involved in the thrombin-induced activation of energy metabolism remain unclear. Here we report an integral analysis on the role of both energy pathways in human platelets activated by several agonists, and the signal transducing mechanisms associated with such activation. We found that thrombin, Trap-6, arachidonic acid, collagen, A23187, epinephrine and ADP significantly increased glycolytic flux (3–38 times <i>vs</i>. non-activated platelets) whereas ristocetin was ineffective. OxPhos (33 times) and mitochondrial transmembrane potential (88%) were increased only by thrombin. OxPhos was the main source of ATP in thrombin-activated platelets, whereas in platelets activated by any of the other agonists, glycolysis was the principal ATP supplier. In order to establish the biochemical mechanisms involved in the thrombin-induced OxPhos activation in platelets, several signaling pathways associated with mitochondrial activation were analyzed. Wortmannin and LY294002 (PI3K/Akt pathway inhibitors), ristocetin and heparin (GPIb inhibitors) as well as resveratrol, ATP (calcium-release inhibitors) and PP1 (Tyr-phosphorylation inhibitor) prevented the thrombin-induced platelet activation. These results suggest that thrombin activates OxPhos and glycolysis through GPIb-dependent signaling involving PI3K and Akt activation, calcium mobilization and protein phosphorylation.</p></div

The impact of self-reported exposure to whole-body-vibrations on the risk of disability pension among men: a 15 year prospective study

<p>Abstract</p> <p>Background</p> <p>Whole-body-vibrations are often associated with adverse health effect but the long term effects are less known. This study investigates the association between occupational exposures to whole-body vibrations, and subsequent transition to disability pension.</p> <p>Methods</p> <p>A total of 4215 male employees were followed up for subsequent disability pension retirement. Exposure to whole-body-vibration was self-reported while new cases of disability pension were retrieved from a national register.</p> <p>Results</p> <p>The hazard ratio (HR) for disability pension retirement among men exposed to whole-body-vibrations was 1.61 (95% confidence interval (CI) 1.07-2.40) after adjustment for age, smoking habits, BMI, physical job demands and awkward work postures. In our model, with the available explanatory variables, 5.6% of the male disability pension cases were attributable to whole-body-vibrations.</p> <p>Conclusions</p> <p>Exposure to whole-body-vibrations predicts subsequent disability pension retirement. Continued reduction of whole-body-vibrations may reduce the number of new cases of disability pension.</p

VIP Regulates the Development & Proliferation of Treg in vivo in spleen

<p>Abstract</p> <p>Background</p> <p>Mounting evidence supports a key role for VIP as an anti-inflammatory agent and promoter of immune tolerance. It suppresses TNF-α and other inflammatory cytokines and chemokines, upregulates anti-inflammatory IL-10, and promotes immune tolerant cells called T regulatory (Treg) cells. VIP KO mice have recently been demonstrated to have spontaneous airway and pulmonary perivascular inflammatory responses, as part of asthma-like and pulmonary hypertension phenotypes, respectively. Both inflammatory responses are correctable with VIP. Focusing on this model, we have now investigated the influence of VIP not only on inflammatory cells but also on Treg cells.</p> <p>Methods</p> <p>Using flow cytometric analysis, we examined the relative preponderance of CD25+CD4+ cells and anti-inflammatory Treg cells, in extracts of thymus and spleen from VIP KO mice (5 VIP KO; 5 VIP KO+ VIP; 10 wild-type). This method allowed antibody-based flow cytometric identification of Treg cells using surface markers CD25 and CD4, along with the: 1) intracellular activation marker FoxP3; and 2) Helios, which distinguishes cells of thymic versus splenic derivation.</p> <p>Conclusions</p> <p>Deletion of the VIP gene results in: 1) CD25+CD4- cell accumulation in the thymus, which is corrected by VIP treatment; 2) more Treg in thymus lacking Foxp3 expression, suggesting VIP is necessary for immune tolerance; and, 3) a tendency towards deficiency of Treg cells in the spleen, which is normalized by VIP treatment. Treg lacking Helios are induced by VIP intrasplenically rather than by migration from the thymus. These results confirm the dual role of VIP as an anti-inflammatory and immune tolerance-promoting agent.</p

De Novo Mutations in PDE10A Cause Childhood-Onset Chorea with Bilateral Striatal Lesions.

Chorea is a hyperkinetic movement disorder resulting from dysfunction of striatal medium spiny neurons (MSNs), which form the main output projections from the basal ganglia. Here, we used whole-exome sequencing to unravel the underlying genetic cause in three unrelated individuals with a very similar and unique clinical presentation of childhood-onset chorea and characteristic brain MRI showing symmetrical bilateral striatal lesions. All individuals were identified to carry a de novo heterozygous mutation in PDE10A (c.898T>C [p.Phe300Leu] in two individuals and c.1000T>C [p.Phe334Leu] in one individual), encoding a phosphodiesterase highly and selectively present in MSNs. PDE10A contributes to the regulation of the intracellular levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Both substitutions affect highly conserved amino acids located in the regulatory GAF-B domain, which, by binding to cAMP, stimulates the activity of the PDE10A catalytic domain. In silico modeling showed that the altered residues are located deep in the binding pocket, where they are likely to alter cAMP binding properties. In vitro functional studies showed that neither substitution affects the basal PDE10A activity, but they severely disrupt the stimulatory effect mediated by cAMP binding to the GAF-B domain. The identification of PDE10A mutations as a cause of chorea further motivates the study of cAMP signaling in MSNs and highlights the crucial role of striatal cAMP signaling in the regulation of basal ganglia circuitry. Pharmacological modulation of this pathway could offer promising etiologically targeted treatments for chorea and other hyperkinetic movement disorders

Gene-enhanced tissue engineering for dental hard tissue regeneration: (2) dentin-pulp and periodontal regeneration

Potential applications for gene-based tissue engineering therapies in the oral and maxillofacial complex include the delivery of growth factors for periodontal regeneration, pulp capping/dentin regeneration, and bone grafting of large osseous defects in dental and craniofacial reconstruction. Part 1 reviewed the principals of gene-enhanced tissue engineering and the techniques of introducing DNA into cells. This manuscript will review recent advances in gene-based therapies for dental hard tissue regeneration, specifically as it pertains to dentin regeneration/pulp capping and periodontal regeneration

Pharmacodynamic evaluation of commonly prescribed oral antibiotics against respiratory bacterial pathogens

<p>Abstract</p> <p>Background</p> <p>Upper and lower respiratory tract infections (RTIs) account for a substantial portion of outpatient antibiotic utilization. However, the pharmacodynamic activity of commonly used oral antibiotic regimens has not been studied against clinically relevant pathogens. The objective of this study was to assess the probability of achieving the requisite pharmacodynamic exposure for oral antibacterial regimens commonly prescribed for RTIs in adults against bacterial isolates frequently involved in these processes (<it>S. pneumoniae</it>, <it>H. influenzae</it>, and <it>M. catharralis</it>).</p> <p>Methods</p> <p>Using a 5000-subject Monte Carlo simulation, the cumulative fractions of response (CFR), (i.e., probabilities of achieving requisite pharmacodynamic targets) for the most commonly prescribed oral antibiotic regimens, as determined by a structured survey of medical prescription patterns, were assessed against local respiratory bacterial isolates from adults in São Paulo collected during the same time period. Minimal inhibitory concentration (MIC) of 230 isolates of <it>Streptococcus pneumoniae </it>(103), <it>Haemophilus influenzae </it>(98), and <it>Moraxella catharralis </it>(29) from a previous local surveillance were used.</p> <p>Results</p> <p>The most commonly prescribed antibiotic regimens were azithromycin 500 mg QD, amoxicillin 500 mg TID, and levofloxacin 500 mg QD, accounting for 58% of the prescriptions. Varied doses of these agents, plus gatifloxacin, amoxicillin-clavulanate, moxifloxacin, and cefaclor made up the remaining regimens. Utilizing aggressive pharmacodynamic exposure targets, the only regimens to achieve greater than 90% CFR against all three pathogens were amoxicillin/amoxicillin-clavulanate 500 mg TID (> 91%), gatifloxacin 400 mg QD (100%), and moxifloxacin 400 mg QD (100%). Considering <it>S. pneumoniae </it>isolates alone, azithromycin 1000 mg QD also achieved greater than 90% CFR (91.3%).</p> <p>Conclusions</p> <p>The only regimens to achieve high CFR against all three pathogen populations in both scenarios were gatifloxacin 400 mg QD, moxifloxacin 400 mg QD, and amoxicillin-clavulanate 500 mg TID. These data suggest the need for reconsideration of empiric antibiotic regimen selection among adult patients with RTIs in the São Paulo area. Additionally, this type of study could be used to optimize prescribing patterns in specific regions in light of emerging resistance.</p

Salvianolic Acid B Prevents Bone Loss in Prednisone-Treated Rats through Stimulation of Osteogenesis and Bone Marrow Angiogenesis

Glucocorticoid (GC) induced osteoporosis (GIO) is caused by the long-term use of GC for treatment of autoimmune and inflammatory diseases. The GC related disruption of bone marrow microcirculation and increased adipogenesis contribute to GIO development. However, neither currently available anti-osteoporosis agent is completely addressed to microcirculation and bone marrow adipogenesis. Salvianolic acid B (Sal B) is a polyphenolic compound from a Chinese herbal medicine, Salvia miltiorrhiza Bunge. The aim of this study was to determine the effects of Sal B on osteoblast bone formation, angiogenesis and adipogenesis-associated GIO by performing marrow adipogenesis and microcirculation dilation and bone histomorphometry analyses. (1) In vivo study: Bone loss in GC treated rats was confirmed by significantly decreased BMD, bone strength, cancellous bone mass and architecture, osteoblast distribution, bone formation, marrow microvessel density and diameter along with down-regulation of marrow BMPs expression and increased adipogenesis. Daily treatment with Sal B (40 mg/kg/d) for 12 weeks in GC male rats prevented GC-induced cancellous bone loss and increased adipogenesis while increasing cancellous bone formation rate with improved local microcirculation by capillary dilation. Treatment with Sal B at a higher dose (80 mg/kg/d) not only prevented GC-induced osteopenia, but also increased cancellous bone mass and thickness, associated with increase of marrow BMPs expression, inhibited adipogenesis and further increased microvessel diameters. (2) In vitro study: In concentration from 10−6 mol/L to 10−7 mol/L, Sal B stimulated bone marrow stromal cell (MSC) differentiation to osteoblast and increased osteoblast activities, decreased GC associated adipogenic differentiation by down-regulation of PPARγ mRNA expression, increased Runx2 mRNA expression without osteoblast inducement, and, furthermore, Sal B decreased Dickkopf-1 and increased β-catenin mRNA expression with or without adipocyte inducement in MSC. We conclude that Sal B prevented bone loss in GC-treated rats through stimulation of osteogenesis, bone marrow angiogenesis and inhibition of adipogenesis

Cthrc1 Is a Positive Regulator of Osteoblastic Bone Formation

Bone mass is maintained by continuous remodeling through repeated cycles of bone resorption by osteoclasts and bone formation by osteoblasts. This remodeling process is regulated by many systemic and local factors.We identified collagen triple helix repeat containing-1 (Cthrc1) as a downstream target of bone morphogenetic protein-2 (BMP2) in osteochondroprogenitor-like cells by PCR-based suppression subtractive hybridization followed by differential hybridization, and found that Cthrc1 was expressed in bone tissues in vivo. To investigate the role of Cthrc1 in bone, we generated Cthrc1-null mice and transgenic mice which overexpress Cthrc1 in osteoblasts (Cthrc1 transgenic mice). Microcomputed tomography (micro-CT) and bone histomorphometry analyses showed that Cthrc1-null mice displayed low bone mass as a result of decreased osteoblastic bone formation, whereas Cthrc1 transgenic mice displayed high bone mass by increase in osteoblastic bone formation. Osteoblast number was decreased in Cthrc1-null mice, and increased in Cthrc1 transgenic mice, respectively, while osteoclast number had no change in both mutant mice. In vitro, colony-forming unit (CFU) assays in bone marrow cells harvested from Cthrc1-null mice or Cthrc1 transgenic mice revealed that Cthrc1 stimulated differentiation and mineralization of osteoprogenitor cells. Expression levels of osteoblast specific genes, ALP, Col1a1, and Osteocalcin, in primary osteoblasts were decreased in Cthrc1-null mice and increased in Cthrc1 transgenic mice, respectively. Furthermore, BrdU incorporation assays showed that Cthrc1 accelerated osteoblast proliferation in vitro and in vivo. In addition, overexpression of Cthrc1 in the transgenic mice attenuated ovariectomy-induced bone loss.Our results indicate that Cthrc1 increases bone mass as a positive regulator of osteoblastic bone formation and offers an anabolic approach for the treatment of osteoporosis

Tinnitus in elderly patients and prognosis of mild-to-moderate congestive heart failure: a cross-sectional study with a long-term extension of the clinical follow-up

<p>Abstract</p> <p>Background</p> <p>The complex mechanism responsible for tinnitus, a symptom highly prevalent in elderly patients, could involve an impaired control of the microcirculation of the inner ear, particularly in patients with poor blood pressure control and impaired left ventricular (LV) function.</p> <p>Methods</p> <p>In order to define the relationship between the presence of tinnitus and the severity and clinical prognosis of mild-to-moderate chronic heart failure (CHF) in a large population of elderly patients (N = 958), a cross-sectional study was conducted with a long-term extension of the clinical follow-up. Blood pressure, echocardiographic parameters, brain natriuretic peptide (BNP), hospitalization, and mortality for CHF were measured. Multivariate logistic regression analysis was used to assess the association between the presence of tinnitus and some of the prognostic determinants of heart failure.</p> <p>Results</p> <p>The presence of tinnitus was ascertained in 233 patients (24.3%; mean age 74.9 ± 6 years) and was associated with reduced systolic and diastolic blood pressure (123.1 ± 16/67.8 ± 9 vs 125.9 ± 15/69.7 ± 9; <it>P </it>= .027/<it>P </it>= .006), reduced LV ejection fraction (LVEF%; 43.6 ± 15 vs 47.9 ± 14%, <it>P </it>= .001), and increased BNP plasma levels (413.1 ± 480 vs 286.2 ± 357, <it>P </it>= .013) in comparison to patients without symptoms. The distribution of CHF functional class was shifted toward a greater severity of the disease in patients with tinnitus. Combined one-year mortality and hospitalization for CHF (events/year) was 1.43 ± 0.2 in patients with tinnitus and 0.83 ± 0.1 in patients without tinnitus, with an adjusted hazard ratio (HR) of 0.61 (95% confidence interval (CI): 0.37 to 0.93, <it>P </it><.002).</p> <p>Conclusions</p> <p>Our preliminary data indirectly support the hypothesis that tinnitus is associated with a worse CHF control in elderly patients and can have some important clinical implications for the early identification of patients who deserve a more aggressive management of CHF.</p