Abnormalities of AMPK Activation and Glucose Uptake in Cultured Skeletal Muscle Cells from Individuals with Chronic Fatigue Syndrome

Background

Post exertional muscle fatigue is a key feature in Chronic Fatigue Syndrome (CFS). Abnormalities of skeletal muscle function have been identified in some but not all patients with CFS. To try to limit potential confounders that might contribute to this clinical heterogeneity, we developed a novel in vitro system that allows comparison of AMP kinase (AMPK) activation and metabolic responses to exercise in cultured skeletal muscle cells from CFS patients and control subjects.

Methods

Skeletal muscle cell cultures were established from 10 subjects with CFS and 7 age-matched controls, subjected to electrical pulse stimulation (EPS) for up to 24h and examined for changes associated with exercise.

Results

In the basal state, CFS cultures showed increased myogenin expression but decreased IL6 secretion during differentiation compared with control cultures. Control cultures subjected to 16h EPS showed a significant increase in both AMPK phosphorylation and glucose uptake compared with unstimulated cells. In contrast, CFS cultures showed no increase in AMPK phosphorylation or glucose uptake after 16h EPS. However, glucose uptake remained responsive to insulin in the CFS cells pointing to an exercise-related defect. IL6 secretion in response to EPS was significantly reduced in CFS compared with control cultures at all time points measured.

Conclusion

EPS is an effective model for eliciting muscle contraction and the metabolic changes associated with exercise in cultured skeletal muscle cells. We found four main differences in cultured skeletal muscle cells from subjects with CFS; increased myogenin expression in the basal state, impaired activation of AMPK, impaired stimulation of glucose uptake and diminished release of IL6. The retention of these differences in cultured muscle cells from CFS subjects points to a genetic/epigenetic mechanism, and provides a system to identify novel therapeutic targets.     

Negative Impact of Skeletal Muscle Loss after Systemic Chemotherapy in Patients with Unresectable Colorectal Cancer

Background

Skeletal muscle depletion (sarcopenia) is closely associated with limited physical ability and high mortality. This study evaluated the prognostic significance of skeletal muscle status before and after chemotherapy in patients with unresectable colorectal cancer (CRC).

Methods

We conducted a retrospective analysis of 215 consecutive patients with unresectable CRC who underwent systemic chemotherapy. Skeletal muscle cross-sectional area was measured by computed tomography. We evaluated the prognostic value of skeletal muscle mass before chemotherapy and the rate of skeletal muscle change in cross-sectional area after chemotherapy.

Results

One-hundred-eighty-two patients met our inclusion criteria. There were no significant differences in progression-free survival (PFS) or overall survival (OS) associated with skeletal muscle mass before chemotherapy. However, 22 patients with skeletal muscle loss (>5%) after chemotherapy showed significantly shorter PFS and OS compared with those without skeletal muscle loss (PFS, log-rank p = 0.029; OS, log-rank p = 0.009). Multivariate Cox regression analysis revealed that skeletal muscle loss after chemotherapy (hazard ratio, 2.079; 95% confidence interval, 1.194–3.619; p = 0.010) was independently associated with OS.

Conclusions

Skeletal muscle loss after chemotherapy was an independent, negative prognostic factor in unresectable CRC.     

Pharmacological targeting of the ephrin receptor kinase signalling by GLPG1790 in vitro and in vivo reverts oncophenotype,induces myogenic differentiation and radiosensitizes embryonal rhabdomyosarcoma cells

Background

EPH (erythropoietin-producing hepatocellular) receptors are clinically relevant targets in several malignancies. This report describes the effects of GLPG1790, a new potent pan-EPH inhibitor, in human embryonal rhabdomyosarcoma (ERMS) cell lines.

Methods

EPH-A2 and Ephrin-A1 mRNA expression was quantified by real-time PCR in 14 ERMS tumour samples and in normal skeletal muscle (NSM). GLPG1790 effects were tested in RD and TE671 cell lines, two in vitro models of ERMS, by performing flow cytometry analysis, Western blotting and immunofluorescence experiments. RNA interfering experiments were performed to assess the role of specific EPH receptors. Radiations were delivered using an x-6 MV photon linear accelerator. GLPG1790 (30 mg/kg) in vivo activity alone or in combination with irradiation (2 Gy) was determined in murine xenografts.

Results

Our study showed, for the first time, a significant upregulation of EPH-A2 receptor and Ephrin-A1 ligand in ERMS primary biopsies in comparison to NSM. GLPG1790 in vitro induced G1-growth arrest as demonstrated by Rb, Cyclin A and Cyclin B1 decrease, as well as by p21 and p27 increment. GLPG1790 reduced migratory capacity and clonogenic potential of ERMS cells, prevented rhabdosphere formation and downregulated CD133, CXCR4 and Nanog stem cell markers. Drug treatment committed ERMS cells towards skeletal muscle differentiation by inducing a myogenic-like phenotype and increasing MYOD1, Myogenin and MyHC levels. Furthermore, GLPG1790 significantly radiosensitized ERMS cells by impairing the DNA double-strand break repair pathway. Silencing of both EPH-A2 and EPH-B2, two receptors preferentially targeted by GLPG1790, closely matched the effects of the EPH pharmacological inhibition. GLPG1790 and radiation combined treatments reduced tumour mass by 83% in mouse TE671 xenografts.

Conclusions

Taken together, our data suggest that altered EPH signalling plays a key role in ERMS development and that its pharmacological inhibition might represent a potential therapeutic strategy to impair stemness and to rescue myogenic program in ERMS cells.

     

Epinephrine Improves the Efficacy of Nebulized Hypertonic Saline in Moderate Bronchiolitis: A Randomised Clinical Trial

Background and Aims

There is no evidence that the epinephrine-3% hypertonic saline combination is more effective than 3% hypertonic saline alone for treating infants hospitalized with acute bronchiolitis. We evaluated the efficacy of nebulized epinephrine in 3% hypertonic saline.

Patients and Methods

We performed a randomized, double-blind, placebo-controlled clinical trial in 208 infants hospitalized with acute moderate bronchiolitis. Infants were randomly assigned to receive nebulized 3% hypertonic saline with either 3 mL of epinephrine or 3 mL of placebo, administered every four hours. The primary outcome measure was the length of hospital stay.

Results

A total of 185 infants were analyzed: 94 in the epinephrine plus 3% hypertonic saline group and 91 in the placebo plus 3% hypertonic saline group. Baseline demographic and clinical characteristics were similar in both groups. Length of hospital stay was significantly reduced in the epinephrine group as compared with the placebo group (3.94 ±1.88 days vs. 4.82 ±2.30 days, P = 0.011). Disease severity also decreased significantly earlier in the epinephrine group (P = 0.029 and P = 0.036 on days 3 and 5, respectively).

Conclusions

In our setting, nebulized epinephrine in 3% hypertonic saline significantly shortens hospital stay in hospitalized infants with acute moderate bronchiolitis compared to 3% hypertonic saline alone, and improves the clinical scores of severity from the third day of treatment, but not before.

Trial Registration

EudraCT 2009-016042-57     

Cigarette Smoke Causes Caspase-Independent Apoptosis of Bronchial Epithelial Cells from Asthmatic Donors

Background

Epidemiologic studies have demonstrated important links between air pollution and asthma. Amongst these pollutants, environmental cigarette smoke is a risk factor both for asthma pathogenesis and exacerbation. As the barrier to the inhaled environment, the bronchial epithelium is a key structure that is exposed to cigarette smoke.

Objectives

Since primary bronchial epithelial cells (PBECs) from asthmatic donors are more susceptible to oxidant-induced apoptosis, we hypothesized that they would be susceptible to cigarette smoke-induced cell death.

Methods

PBECs from normal and asthmatic donors were exposed to cigarette smoke extract (CSE); cell survival and apoptosis were assessed by fluorescence-activated cell sorting, and protective effects of antioxidants evaluated. The mechanism of cell death was evaluated using caspase inhibitors and immunofluorescent staining for apoptosis-inducing factor (AIF).

Results

Exposure of PBEC cultures to CSE resulted in a dose-dependent increase in cell death. At 20% CSE, PBECs from asthmatic donors exhibited significantly more apoptosis than cells from non-asthmatic controls. Reduced glutathione (GSH), but not ascorbic acid (AA), protected against CSE-induced apoptosis. To investigate mechanisms of CSE-induced apoptosis, caspase-3 or -9 inhibitors were tested, but these failed to prevent apoptosis; in contrast, CSE promoted nuclear translocation of AIF from the mitochondria. GSH reduced the number of nuclear-AIF positive cells whereas AA was ineffective.

Conclusion

Our results show that PBECs from asthmatic donors are more susceptible to CSE-induced apoptosis. This response involves AIF, which has been implicated in DNA damage and ROS-mediated cell-death. Epithelial susceptibility to CSE may contribute to the impact of environmental tobacco smoke in asthma.     

RNA Interference of GADD153 Protects Photoreceptors from Endoplasmic Reticulum Stress-Mediated Apoptosis after Retinal Detachment

Background

Apoptosis of photoreceptors plays a critical role in the vision loss caused by retinal detachment (RD). Pharmacologic inhibition of photoreceptor cell death may prevent RD. This study investigated the role of GADD153 that participates in endoplasmic reticulum (ER) stress-mediated apoptosis of photoreceptor cells after RD.

Methods

Retinal detachment was created in Wistar rats by subretinal injection of hyaluronic acid. The rats were then randomly divided into four groups: normal control group, RD group, GADD153 RNAi group and vehicle group. RNA interference of GADD153 was performed using short hairpin RNA (shRNA). Expressions of GADD153 mRNA and protein were examined by RT-PCR and Western blotting analysis, respectively. GADD153 protein distribution in the retinal cells was observed using immunofluorescence confocal laser scanning microscopy. Apoptosis of retinal cells was determined by TdT-mediated fluorescein-16-dUTP nick-end labeling (TUNEL) assay.

Results

Lentivirus GADD153 shRNA with the most effective silencing effect was chosen for in vivo animal study and was successfully delivered into the retinal tissues. GADD153 mRNA and protein expressions in GADD153 RNAi group were significantly lower than those in the RD group. Silencing of GADD153 by RNAi protected photoreceptors from ER stress-induced apoptosis.

Conclusion

ER stress-mediated pathway is involved in photoreceptor cell apoptosis after RD. GADD153 is a key regulatory molecule regulating ER-stress pathways and plays a crucial role in the apoptosis of photoreceptor cells after RD.     

Center of Pressure Displacement of Standing Posture during Rapid Movements Is Reorganised Due to Experimental Lower Extremity Muscle Pain

Background

Postural control during rapid movements may be impaired due to musculoskeletal pain. The purpose of this study was to investigate the effect of experimental knee-related muscle pain on the center of pressure (CoP) displacement in a reaction time task condition.

Methods

Nine healthy males performed two reaction time tasks (dominant side shoulder flexion and bilateral heel lift) before, during, and after experimental pain induced in the dominant side vastus medialis or the tibialis anterior muscles by hypertonic saline injections. The CoP displacement was extracted from the ipsilateral and contralateral side by two force plates and the net CoP displacement was calculated.

Results

Compared with non-painful sessions, tibialis anterior muscle pain during the peak and peak-to-peak displacement for the CoP during anticipatory postural adjustments (APAs) of the shoulder task reduced the peak-to-peak displacement of the net CoP in the medial-lateral direction (P<0.05). Tibialis anterior and vastus medialis muscle pain during shoulder flexion task reduced the anterior-posterior peak-to-peak displacement in the ipsilateral side (P<0.05).

Conclusions

The central nervous system in healthy individuals was sufficiently robust in maintaining the APA characteristics during pain, although the displacement of net and ipsilateral CoP in the medial-lateral and anterior-posterior directions during unilateral fast shoulder movement was altered.     

Magnetic Resonance Imaging Is Sensitive to Pathological Amelioration in a Model for Laminin-Deficient Congenital Muscular Dystrophy (MDC1A)

Purpose

To elucidate the reliability of MRI as a non-invasive tool for assessing in vivo muscle health and pathological amelioration in response to Losartan (Angiotensin II Type 1 receptor blocker) in DyW mice (mouse model for Laminin-deficient Congenital Muscular Dystrophy Type 1A).

Methods

Multiparametric MR quantifications along with histological/biochemical analyses were utilized to measure muscle volume and composition in untreated and Losartan-treated 7-week old DyW mice.

Results

MRI shows that DyW mice have significantly less hind limb muscle volume and areas of hyperintensity that are absent in WT muscle. DyW mice also have significantly elevated muscle levels (suggestive of inflammation and edema). Muscle T₂ returned to WT levels in response to Losartan treatment. When considering only muscle pixels without T₂ elevation, DyW T₂ levels are significantly lower than WT (suggestive of fibrosis) whereas Losartan-treated animals do not demonstrate this decrease in muscle T₂. MRI measurements suggestive of elevated inflammation and fibrosis corroborate with increased Mac-1 positive cells as well as increased Picrosirius red staining/COL1a gene expression that is returned to WT levels in response to Losartan.

Conclusions

MRI is sensitive to and tightly corresponds with pathological changes in DyW mice and thus is a viable and effective non-invasive tool for assessing pathological changes.     

Polyunsaturated Fatty Acids Attenuate Diet Induced Obesity and Insulin Resistance,Modulating Mitochondrial Respiratory Uncoupling in Rat Skeletal Muscle

Objectives

Omega (ω)-3 polyunsaturated fatty acids (PUFA) are dietary compounds able to attenuate insulin resistance. Anyway, the precise actions of ω-3PUFAs in skeletal muscle are overlooked. We hypothesized that PUFAs, modulating mitochondrial function and efficiency, would ameliorate pro-inflammatory and pro-oxidant signs of nutritionally induced obesity.

Study Design

To this aim, rats were fed a control diet (CD) or isocaloric high fat diets containing either ω-3 PUFA (FD) or lard (LD) for 6 weeks.

Results

FD rats showed lower weight, lipid gain and energy efficiency compared to LD-fed animals, showing higher energy expenditure and O₂ consumption/CO₂ production. Serum lipid profile and pro-inflammatory parameters in FD-fed animals were reduced compared to LD. Accordingly, FD rats exhibited a higher glucose tolerance revealed by an improved glucose and insulin tolerance tests compared to LD, accompanied by a restoration of insulin signalling in skeletal muscle. PUFAs increased lipid oxidation and reduced energy efficiency in subsarcolemmal mitochondria, and increase AMPK activation, reducing both endoplasmic reticulum and oxidative stress. Increased mitochondrial respiration was related to an increased mitochondriogenesis in FD skeletal muscle, as shown by the increase in PGC1-α and -β.

Conclusions

our data strengthened the association of high dietary ω3-PUFA intake with reduced mitochondrial energy efficiency in the skeletal muscle.     

Morphological Changes of Human Corneal Endothelial Cells after Rho-Associated Kinase Inhibitor Eye Drop (Ripasudil) Administration: A Prospective Open-Label Clinical Study

Purpose

To investigate the effect and safety of a selective Rho kinase inhibitor, ripasudil 0.4% eye drops, on corneal endothelial cells of healthy subjects.

Design

Prospective, interventional case series.

Methods

In this study, 6 healthy subjects were administered ripasudil 0.4% in the right eye twice daily for 1 week. Morphological changes and corneal endothelial cell density were examined by noncontact and contact specular microscopy. Central corneal thickness and corneal volume of 5 mm-diameter area of center cornea were analyzed by Pentacam Scheimpflug topography. All the above measurements were conducted in both eyes before administration, 1.5 and 6 hours after the initial administration on day 0; and in the same manner after the final administration on day 7.

Results

By noncontact specular microscopy, indistinct cell borders with pseudo guttae were observed, but by contact specular microscopy, morphological changes of corneal endothelial cells were mild and pseudo guttae was not observed after single and repeated administration of ripasudil in all subjects. These changes resolved prior to the next administration, and corneal endothelial cell density, central corneal thickness and corneal volume were not changed throughout the study period.

Conclusion

Transient morphological changes of corneal endothelial cells such as indistinct cell borders with pseudo guttae were observed by noncontact specular microscopy in healthy subjects after ripasudil administration. Corneal edema was not observed and corneal endothelial cell density did not decrease after 1 week repetitive administration. These morphological changes were reversible and corneal endothelial cell morphology returned to normal prior to the next administration.

Trial Registration

JAPIC Clinical Trials Information 142705     

Nox4 Is Dispensable for Exercise Induced Muscle Fibre Switch

Introduction

By producing H₂O₂, the NADPH oxidase Nox4 is involved in differentiation of mesenchymal cells. Exercise alters the composition of slow and fast twitch fibres in skeletal. Here we hypothesized that Nox4 contributes to exercise-induced adaptation such as changes in muscle metabolism or muscle fibre specification and studied this in wildtype and Nox4-/- mice.

Results

Exercise, as induced by voluntary running in a running wheel or forced running on a treadmill induced a switch from fast twitch to intermediate fibres. However the induced muscle fibre switch was similar between Nox4-/- and wildtype mice. The same held true for exercise-induced expression of PGC1α or AMPK activation. Both are increased in response to exercise, but with no difference was observed between wildtype and Nox4-/- mice.

Conclusion

Thus, exercise-induced muscle fibre switch is Nox4-independent.     

Expression of Toll-Like Receptor -7 and -9 in B Cell Subsets from Patients with Primary Sj?gren’s Syndrome

Introduction

Sjögren’s syndrome (SS) is a rheumatic autoimmune disease characterized by inflammation of exocrine glands. As autoantibodies are present in a majority of patients, B cells have been suggested to play an important role in onset and development of the disease. Toll-like receptors (TLRs) are pattern recognition receptors triggering innate immune responses. Since an increased expression of TLRs has been detected in other rheumatic diseases the purpose of this study was to explore TLRs in B cells of SS patients.

Methods

The expression of TLR-7 and -9 in B cell subsets of 25 patients with primary SS (pSS) and 25 healthy controls was analysed in peripheral blood using flow cytometry and real time quantitative PCR.

Results

We detected similar levels of CD19⁺ B cells in pSS patients and healthy controls. An increased number of naïve B cells, as well as fewer pre-switched memory B cells were found in pSS patients. No significant differences were observed in TLR-7 and -9 expression in B cells between pSS patients and healthy controls.

Conclusion

This study shows that pSS patients have an alteration in the B cell subpopulation composition compared to controls, with less pre-switched memory B cells and more naïve B cells. We did not detect any significant disparities in TLR-7 and -9 expression between the two groups.     

Sternal Wound Infection after Cardiac Surgery: Management and Outcome

Background

Sternal Wound Infection (SWI) is a severe complication after cardiac surgery. Debridement associated with primary closure using Redon drains (RD) is an effective treatment, but data on RD management and antibiotic treatment are scarce.

Methods

We performed a single-center analysis of consecutive patients who were re-operated for SWI between 01/2009 and 12/2012. All patients underwent a closed drainage with RD (CDRD). Patients with endocarditis or those who died within the first 45 days were excluded from management analysis. RD fluid was cultured twice weekly. Variables recorded were clinical and biological data at SWI diagnosis, severity of SWI based on criteria for mediastinitis as defined by the Centers for Disease Control (CDC), antibiotic therapy, RD management and patient’s outcome.

Results

160 patients developed SWI, 102 (64%) fulfilled CDC criteria (CDC+) and 58 (36%) did not (CDC- SWI). Initial antibiotic treatment and surgical management were similar in CDC+ and CDC- SWI. Patients with CDC+ SWI had a longer duration of antibiotic therapy and a mortality rate of 17% as compared to 3% in patients with CDC- SWI (p = 0.025). Rates of superinfection (10% and 9%) and need for second reoperation (12% and 17%) were similar. Failure (death or need for another reoperation) was associated with female gender, higher EuroScore for prediction of operative mortality, and stay in the ICU.

Conclusion

In patients with SWI, initial one-stage surgical debridement with CDRD is associated with favorable outcomes. CDC+ and CDC- SWI received essentially the same management, but CDC+ SWI has a more severe outcome.     

Tuberculosis Case Fatality and Other Causes of Death among Multidrug-Resistant Tuberculosis Patients in a High HIV Prevalence Setting, 2000-2008, South Africa

Introduction

South Africa has the highest reported rates of multi-drug resistant TB in Africa, typified by poor treatment outcomes, attributable mainly to high default and death rates. Concomitant HIV has become the strongest predictor of death among MDR-TB patients, while anti-retroviral therapy (ART) has dramatically reduced mortality. TB Case fatality rate (CFR) is an indicator that specifically reports on deaths due to TB.

Aim

The aim of this paper was to investigate causes of death amongst MDR-TB patients, the contribution of conditions other than TB to deaths, and to determine if causes differ between HIV-uninfected patients, HIV-infected patients receiving ART and those without ART.

Methods

We carried out a retrospective review of data captured from the register of the MDR-TB programme of the North West Province, South Africa. We included 671 patients treated between 2000–2008; 59% of the cohort was HIV-infected and 33% had received ART during MDR treatment. The register contained data on treatment outcomes and causes of death.

Results

Treatment outcomes between HIV-uninfected cases, HIV-infected cases receiving ART and HIV-infected without ART differed significantly (p<0.000). The cohort death rate was 24%, 13% for HIV-uninfected cases and 31% for HIV-infected cases. TB caused most of the deaths, resulting in a cohort CFR of 15%, 9% for HIV-uninfected cases and 20% for HIV-infected cases. Cohort mortality rate due to other conditions was 2%. AIDS-conditions rather than TB caused significantly more deaths among HIV-infected cases receiving ART than those not (p = 0.02).

Conclusions

The deaths among HIV-infected individuals contribute substantially to the high death rate. ART co-therapy protected HIV-infected cases from death due to TB and AIDS-conditions. Mechanisms need to be in place to ensure that HIV-infected individuals are retained in care upon completion of their MDR-TB treatment.     

Comparison of Normal Saline,Hypertonic Saline Albumin and Terlipressin plus Hypertonic Saline Albumin in an Infant Animal Model of Hypovolemic Shock

Introduction

In series of cases and animal models suffering hemorrhagic shock, the use of vasopressors has shown potential benefits regarding hemodynamics and tissue perfusion. Terlipressin is an analogue of vasopressin with a longer half-life that can be administered by bolus injection. We have previously observed that hypertonic albumin improves resuscitation following controlled hemorrhage in piglets. The aim of the present study was to analyze whether the treatment with the combination of terlipressin and hypertonic albumin can produce better hemodynamic and tissular perfusion parameters than normal saline or hypertonic albumin alone at early stages of hemorrhagic shock in an infant animal model.

Methods

Experimental, randomized animal study including 39 2-to-3-month-old piglets. Thirty minutes after controlled 30 ml/kg bleed, pigs were randomized to receive either normal saline (NS) 30 ml/kg (n = 13), 5% albumin plus 3% hypertonic saline (AHS) 15 ml/kg (n = 13) or single bolus of terlipressin 15 μg/kg i.v. plus 5% albumin plus 3% hypertonic saline 15 ml/kg (TAHS) (n = 13) over 30 minutes. Global hemodynamic and tissular perfusion parameters were compared.

Results

After controlled bleed a significant decrease of blood pressure, cardiac index, central venous saturation, carotid and peripheral blood flow, brain saturation and an increase of heart rate, gastric PCO₂ and lactate was observed. After treatment no significant differences in most hemodynamic (cardiac index, mean arterial pressure) and perfusion parameters (lactate, gastric PCO₂, brain saturation, cutaneous blood flow) were observed between the three therapeutic groups. AHS and TAHS produced higher increase in stroke volume index and carotid blood flow than NS.

Conclusions

In this pediatric animal model of hypovolemic shock, albumin plus hypertonic saline with or without terlipressin achieved similar hemodynamics and perfusion parameters than twice the volume of NS. Addition of terlipressin did not produce better results than AHS.     

Genotyping on ALDH2: Comparison of Four Different Technologies

Objectives

This study aimed to compare the accuracy and performance of four genotyping methods for detecting single nucleotide polymorphisms (SNPs) in aldehyde dehydrogenase-2 (ALDH2), which is the principal enzyme involved in alcohol metabolism.

Design and Methods

We genotyped rs671 of ALDH2 in 96 coronary heart disease (CHD) patients with four methods including high resolution melting analysis (HRM), TaqMan allelic discrimination assay (TaqMan), allele-specific PCR (AS-PCR) and pyrosequencing. Meanwhile, we compared the accuracy and performance of these methods.

Results

All selected patients were successfully genotyped with referred methods. The results of these four assays showed 100% concordant results and had 100% accuracy as verified by Sanger sequencing.

Conclusions

All of the referred methods can be used for genotyping ALDH2 rs671 with the same accuracy compared to Sanger sequencing. In small size of clinical samples, HRM and AS-PCR outperform over others due to their lower cost and less hands-on operation, which are suitable for clinical application.     

Increase in Red Blood Cell-Nitric Oxide Synthase Dependent Nitric Oxide Production during Red Blood Cell Aging in Health and Disease: A Study on Age Dependent Changes of Rheologic and Enzymatic Properties in Red Blood Cells

Aim

To investigate RBC-NOS dependent NO signaling during in vivo RBC aging in health and disease.

Method

RBC from fifteen healthy volunteers (HC) and four patients with type 2 diabetes mellitus (DM) were separated in seven subpopulations by Percoll density gradient centrifugation.

Results

The proportion of old RBC was significantly higher in DM compared to HC. In both groups, in vivo aging was marked by changes in RBC shape and decreased cell volume. RBC nitrite, as marker for NO, was higher in DM and increased in both HC and DM during aging. RBC deformability was lower in DM and significantly decreased in old compared to young RBC in both HC and DM. RBC-NOS Serine¹¹⁷⁷ phosphorylation, indicating enzyme activation, increased during aging in both HC and DM. Arginase I activity remained unchanged during aging in HC. In DM, arginase I activity was significantly higher in young RBC compared to HC but decreased during aging. In HC, concentration of L-arginine, the substrate of RBC-NOS and arginase I, significantly dropped from young to old RBC. In DM, L-arginine concentration was significantly higher in young RBC compared to HC and significantly decreased during aging. In blood from healthy subjects, RBC-NOS activation was additionally inhibited by N⁵-(1-iminoethyl)-L-Ornithine dihydrochloride which decreased RBC nitrite, and impaired RBC deformability of all but the oldest RBC subpopulation.

Conclusion

This study first-time showed highest RBC-NOS activation and NO production in old RBC, possibly to counteract the negative impact of cell shrinkage on RBC deformability. This was even more pronounced in DM. It is further suggested that highly produced NO only insufficiently affects cell function of old RBC maybe because of isolated RBC-NOS in old RBC thus decreasing NO bioavailability. Thus, increasing NO availability may improve RBC function and may extend cell life span in old RBC.     

Study of muscle cell dedifferentiation after skeletal muscle injury of mice with a Cre-Lox system

Background

Dedifferentiation of muscle cells in the tissue of mammals has yet to be observed. One of the challenges facing the study of skeletal muscle cell dedifferentiation is the availability of a reliable model that can confidentially distinguish differentiated cell populations of myotubes and non-fused mononuclear cells, including stem cells that can coexist within the population of cells being studied.

Methodology/Principal Findings

In the current study, we created a Cre/Lox-β-galactosidase system, which can specifically tag differentiated multinuclear myotubes and myotube-generated mononuclear cells based on the activation of the marker gene, β-galactosidase. By using this system in an adult mouse model, we found that β-galactosidase positive mononuclear cells were generated from β-galactosidase positive multinuclear myofibers upon muscle injury. We also demonstrated that these mononuclear cells can develop into a variety of different muscle cell lineages, i.e., myoblasts, satellite cells, and muscle derived stem cells.

Conclusions/Significance

These novel findings demonstrated, for the first time, that cellular dedifferentiation of skeletal muscle cells actually occurs in mammalian skeletal muscle following traumatic injury in vivo.     

Cost-Effectiveness of Dolutegravir in HIV-1 Treatment-Experienced (TE) Patients in France

Objectives

To evaluate the cost-effectiveness of a new generation integrase inhibitor (INI), dolutegravir (DTG), in France, in treatment-experienced (TE) and INI-naïve HIV-infected adults with at least two classes resistance compared to raltegravir (RAL), by adapting previously published Anti-Retroviral Analysis by Monte Carlo Individual Simulation (ARAMIS) model.

Methods

ARAMIS is a microsimulation Markov model with a lifetime time horizon and a monthly cycle length. Health states are defined as with or without opportunistic infection and death. In the initial cohort, efficacy and safety data were derived from a phase III study comparing DTG to RAL. Antiretroviral treatment algorithms, accounting for patient history, were based on French guidelines and experts opinion. Costs are mainly including treatment costs, routine HIV and opportunistic infection care, and death. Utilities depend on CD4+ cell count and the occurrence of opportunistic infections.

Results

The ARAMIS model indicates in the TE population that DTG compared to RAL over a life time is associated with 0.35 additional quality-adjusted life years (QALY; 10.75 versus 10.41) and additional costs of €7,266 (€390,001 versus €382,735). DTG increased costs are mainly related to a 9.1-month increase in life expectancy for DTG compared with RAL, and consequently a longer time spent on ART. The incremental cost-effectiveness ratio (ICER) for DTG compared with RAL is €21,048 per QALY gained. About 83% and 14% of total lifetime costs are associated with antiretroviral therapy and routine HIV care respectively. Univariate deterministic sensitivity analyses demonstrate the robustness of the model.

Conclusion

DTG is cost-effective in the management of TE INI naive patients in France, from a collective perspective. These results could be explained by the superior efficacy of DTG in this population and its higher genetic barrier to resistance compared to RAL. These data need to be confirmed with longer-term real life data.     

New Insight into the Time-Course of Motor and Sensory System Changes in Pain

Background

Pain-related interactions between primary motor (M1) and primary sensory (S1) cortex are poorly understood. In particular, the time-course over which S1 processing and corticomotor output are altered in association with muscle pain is unclear. We aimed to examine the temporal profile of altered processing in S1 and altered corticomotor output with finer temporal resolution than has been used previously.

Methods

In 10 healthy individuals we recorded somatosensory evoked potentials (SEPs) and motor evoked potentials (MEPs) in separate sessions at multiple time-points before, during and immediately after pain induced by hypertonic saline infusion in a hand muscle, and at 15 and 25 minutes follow-up.

Results

Participants reported an average pain intensity that was less in the session where SEPs were recorded (SEPs: 4.0±1.6; MEPs: 4.9±2.3). In addition, the time taken for pain to return to zero once infusion of hypertonic saline ceased was less for participants in the SEP session (SEPs: 4.7±3.8 mins; MEPs 9.4±7.4 mins). Both SEPs and MEPs began to reduce almost immediately after pain reached 5/10 following hypertonic saline injection and were significantly reduced from baseline by the second (SEPs) and third (MEPs) recording blocks during pain. Both parameters remained suppressed immediately after pain had resolved and at 15 and 25 minutes after the resolution of pain.

Conclusions

These data suggest S1 processing and corticomotor output may be co-modulated in association with muscle pain. Interestingly, this is in contrast to previous observations. This discrepancy may best be explained by an effect of the SEP test stimulus on the corticomotor pathway. This novel finding is critical to consider in experimental design and may be potentially useful to consider as an intervention for the management of pain.