Good manufacturing practice-compliant cell sorting and large-scale expansion of single KIR-positive alloreactive human natural killer cells for multiple infusions to leukemia patients

Background aimsAlloreactive natural killer (NK) cells are potent effectors of innate anti-tumor defense. The introduction of NK cell-based immunotherapy to current treatment options in acute myeloid leukemia (AML) requires NK cell products with high anti-leukemic efficacy optimized for clinical use.MethodsWe describe a good manufacturing practice (GMP)-compliant protocol of large-scale ex vivo expansion of alloreactive NK cells suitable for multiple donor lymphocyte infusions (NK-DLI) in AML. CliniMACS-purified NK cells were cultured in closed air-permeable culture bags with certified culture medium and components approved for human use [human serum, interleukin (IL)-2, IL-15 and anti-CD3 antibody] and with autologous irradiated feeder cells.ResultsNK cells (6.0 ± 1.2 × 10⁸) were purified from leukaphereses (8.1 ± 0.8 L) of six healthy donors and cultured under GMP conditions. NK cell numbers increased 117.0 ± 20.0-fold in 19 days. To reduce the culture volume associated with expansion of bulk NK cells and to expand selectively the alloreactive NK cell subsets, GMP-certified cell sorting was introduced to obtain cells with single killer immunoglobulin-like receptor (KIR) specificities. The subsequent GMP-compliant expansion of single KIR⁺ cells was 268.3 ± 66.8-fold, with a contaminating T-cell content of only 0.006 ± 0.002%. The single KIR-expressing NK cells were cytotoxic against HLA-mismatched primary AML blasts in vitro and effectively reduced tumor cell load in vivo in NOD/SCID mice transplanted with human AML.ConclusionsThe approach to generating large numbers of GMP-grade alloreactive NK cells described here provides the basis for clinical efficacy trials of NK-DLI to complement and advance therapeutic strategies against human AML.     

Local muscle cooling does not impact expression of mitochondrial-related genes

Recovery that takes place in a cold environment after endurance exercise elevates PGC-1α mRNA whereas ERRα and NRF2 mRNA expression are inhibited. However, the effect of local skeletal muscle cooling on mitochondrial-related gene expression is unknown.PurposeTo determine the impact of local skeletal muscle cooling during recovery from an acute bout of exercise on mitochondrial-related gene expression.MethodsRecreationally-trained male cyclists (n=8, age 25±3 y, height 181±6 cm, weight 79±8 kg, 12.8±3.6% body fat, VO_2peak 4.52±0.88 L·min⁻¹ protocol) completed a 90-min variable intensity cycling protocol followed by 4 h of recovery. During recovery, ice was applied intermittently to one leg (ICE) while the other leg served as a control (CON). Intramuscular temperature was recorded continuously. Muscle biopsies were taken from each vastus lateralis at 4 h post-exercise for the analysis of mitochondrial-related gene expression.ResultsIntramuscular temperature was colder in ICE (26.7±1.1 °C) than CON (35.5±0.1 °C) throughout the 4 h recovery period (p<0.001). There were no differences in expression of PGC-1α, TFAM, NRF1, NRF2, or ERRα mRNA between ICE and CON after the 4 h recovery period.ConclusionLocal muscle cooling after exercise does not impact the expression of mitochondrial biogenesis-related genes compared to recovery from exercise in control conditions. When these data are considered with previous research, the stimuli for cold-induced gene expression alterations may be related to factors other than local muscle temperature. Additionally, different intramuscular temperatures should be examined to determine dose-response of mitochondrial-related gene expression.     

The protective effect of montelukast sodium on carbon tetrachloride induced hepatopathy in rat

AimThis study investigates the effects of montelukast sodium (MK) (CysLTLT1 receptor antagonist) on CCl₄induced hepatopathy on rat.Material and methodsWe worked on 4 groups of 10 Wistar male rats each. The groups received as follows: group I (control group) – saline, group II – MK 5 mg/kg/day i.p. for 5 days, group III – MK 5 mg/kg/day i.p., 1 day prior to and 4 days concomitantly with CCl₄ p.o., 0.3 ml/Kg/day and group IV – CCl₄, p.o., 0.3 ml/Kg/day for 4 days. One day after the last administration, samples of blood were taken and alanine aminotransferase (ALT), total bilirubin (TB), direct bilirubin (DB), malondialdehyde (MDA), catalase (CAT) as well as total antioxidant capacity (TAC) were determined. The histopathological exam was performed. We also determined superoxide dismutase (SOD), MDA, CAT and GSH in liver homogenate.ResultsCompared to group IV, group III exhibited statistically significant lower levels of ALT (318 ± 15.75 versus 203.14 ± 10.28 UI, p < 0.0001), TB (3.16 ± 0.30 versus 1.99 ± 0.08 mg/dl, p < 0.0001), MDA in blood and in liver homogenate (4.98 ± 1.71 versus 2.15 ± 1.18 nmol/ml, p = 0.0004) and higher levels of SOD and CAT. Histopathologically, group IV presented important macro- and micro-vesicular hepatic steatosis and group III preserved lobular histoarchitecture and had less severe cellular lesions.ConclusionMK exhibits a partial hepatoprotective effect on rats treated with CCl₄.     

High-dose vitamin C supplementation increases skeletal muscle vitamin C concentration and SVCT2 transporter expression but does not alter redox status in healthy males

Antioxidant vitamin C (VC) supplementation is of potential clinical benefit to individuals with skeletal muscle oxidative stress. However, there is a paucity of data reporting on the bioavailability of high-dose oral VC in human skeletal muscle. We aimed to establish the time course of accumulation of VC in skeletal muscle and plasma during high-dose VC supplementation in healthy individuals. Concurrently we investigated the effects of VC supplementation on expression levels of the key skeletal muscle VC transporter sodium-dependent vitamin C transporter 2 (SVCT2) and intramuscular redox and mitochondrial measures. Eight healthy males completed a randomized placebo-controlled, crossover trial involving supplementation with ascorbic acid (2×500 mg/day) over 42 days. Participants underwent muscle and blood sampling on days 0, 1, 7, and 42 during each treatment. VC supplementation significantly increased skeletal muscle VC concentration after 7 days, which was maintained at 42 days (VC 3.0±0.2 (mean±SEM) to 3.9±0.4 mg/100 g wet weight (ww) versus placebo 3.1±0.3 to 2.9±0.2 mg/100 g ww, p=0.001). Plasma VC increased after 1 day, which was maintained at 42 days (VC 61.0±6.1 to 111.5±10.4 µmol/L versus placebo 60.7±5.3 to 59.2±4.8 µmol/L, p<0.001). VC supplementation significantly increased skeletal muscle SVCT2 protein expression (main treatment effect p=0.006) but did not alter skeletal muscle redox measures or citrate synthase activity. A main finding of our study was that 7 days of high-dose VC supplementation was required to significantly increase skeletal muscle vitamin C concentration in healthy males. Our findings implicate regular high-dose vitamin C supplementation as a means to safely increase skeletal muscle vitamin C concentration without impairing intramuscular ascorbic acid transport, antioxidant concentrations, or citrate synthase activity.     

Effect of pain location on spatial reorganisation of muscle activity

IntroductionWe aimed to determine whether the changes in muscle activity (in terms of both gross electromyography (EMG) and motor unit (MU) discharge characteristics) observed during pain are spatially organized with respect to pain location within a muscle which is the main contributor of the task.MethodsSurface and fine-wire EMG was recorded during matched low-force isometric plantarflexion from soleus (from four quadrants with fine-wire EMG and from the medial/lateral sides with surface EMG), both gastrocnemii heads, peroneus longus, and tibialis anterior. Four conditions were tested: two control conditions that each preceded contractions with pain induced in either the lateral (Pain_L) or medial (Pain_M) side of soleus.ResultsNeither the presence (p = 0.28) nor location (p = 0.19) of pain significantly altered gross muscle activity of any location (lateral/medial side of soleus, gastrocnemii, peroneus longus and tibialis anterior). Group data from 196 MUs show redistribution of MU activity throughout the four quadrants of soleus, irrespective of pain location. The significant decrease of MU discharge rate during pain (p < 0.0001; Pain_L: 7.3 ± 0.9–6.9 ± 1.1 Hz, Pain_M: 7.0 ± 1.1 to 6.6 ± 1.1 Hz) was similar for all quadrants of the soleus (p = 0.43), regardless of the pain location (p = 0.98). There was large inter-participant variation in respect to the characteristics of the altered MU discharge with pain.ConclusionResults from both surface and fine-wire EMG recordings do not support the hypothesis that muscle activity is reorganized in a simple systematic manner with respect to pain location.     

Changes in muscle activation patterns and subjective low back pain ratings during prolonged standing in response to an exercise intervention

BackgroundLow back pain (LBP) development has been associated with occupational standing. Increased hip and trunk muscle co-activation is considered to be predisposing for LBP development during standing in previously asymptomatic individuals. The purpose of this work was to investigate muscle activation and LBP responses to a prescribed exercise program. Pain-developing (PD) individuals were expected to have decreased LBP and muscle co-activation following exercise intervention.MethodsElectromyography (EMG) data were recorded from trunk and hip muscle groups during 2-h of standing. An increase of >10 mm on visual analog scale (VAS) during standing was threshold for PD categorization. Participants were assigned to progressive exercise program with weekly supervision or control (usual activity) for 4 weeks then re-tested.ResultsForty percent were categorized as PD on day 1, VAS = 24.2 (±4.0) mm. PD exercisers (PDEX) had lower VAS scores (8.93 ± 3.66 mm) than PD control (PDCON) (16.5 ± 6.3 mm) on day 2 (p = 0.007). Male PDEX had decreased gluteus medius co-activation levels (p < 0.05) on day 2.DiscussionThe exercise program proved beneficial in reducing LBP during standing. There were changes in muscle activation patterns previously associated with LBP. Predisposing factors for LBP during standing were shown to change positively with appropriate exercise intervention.     

Conduction velocity of the human phrenic nerve in the neck

PurposeTo measure phrenic nerve conduction velocity in the neck in humans.ScopeWe studied 15 healthy subjects (9 men, 32.4 ± 6.7). We performed bipolar electrical phrenic stimulation in the neck, from a distal and a proximal stimulation site, and recorded diaphragm electromyographic responses on the surface of the chest. The ratio of the between-site distance to the latency difference provided phrenic velocities. Ulnar motor velocity was assessed similarly. In addition, five homogeneous patients with Charcot-Marie-Tooth disease type 1A (CMT1A) were studied for validation purposes. We obtained diaphragmatic responses from the two stimulation sites in all cases. The distal latencies (anterior axillary line recording) were 6.51 ± 0.63 ms (right) and 6.13 ± 0.64 ms (left). The minimal between site distance was 39 mm. Phrenic motor velocity was 55.2 ± 6.3 m s^?1 (right) and 56.3 ± 7.2 m s^?1 (left). In CMT1A, phrenic velocities were 17.1 ± 8.1 m s^?1 (from 7 to 32 m s^?1) and were similar to ulnar and median velocities.ConclusionsPhrenic nerve velocities can be estimated in humans and compare with upper limb motor conduction velocities. This should refine the investigation of phrenic function in peripheral neuropathies.     

The relationship between external knee moments and muscle co-activation in subjects with medial knee osteoarthritis

PurposeExternal knee moments are reliable to measure knee load but it does not take into account muscle activity. Considering that muscle co-activation increases compressive forces at the knee joint, identifying relationships between muscle co-activations and knee joint load would complement the investigation of the knee loading in subjects with knee osteoarthritis. The purpose of this study was to identify relationships between muscle co-activation and external knee moments during walking in subjects with medial knee osteoarthritis.Methods19 controls (11 males, aged 56.6 ± 5, and BMI 25.2 ± 3.3) and 25 subjects with medial knee osteoarthritis (12 males, aged 57.3 ± 5.3, and BMI 28.2 ± 4) were included in this study. Knee adduction and flexion moments, and co-activation (ratios and sums of quadriceps, hamstring, and gastrocnemius) were assessed during walking and compared between groups. The relationship between knee moments and co-activation was investigated in both groups.FindingsSubjects with knee osteoarthritis presented a moderate and strong correlation between co-activation (ratios and sums) and knee moments.InterpretationMuscle co-activation should be used to measure the contribution of quadriceps, hamstring, and gastrocnemius on knee loading. This information would cooperate to develop a more comprehensive approach of knee loading in this population.     

The effect of PI3 kinase inhibitor LY294002 on voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

AimsWe examined the effect of LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, on voltage-dependent K⁺ (Kv) channels.Main methodsElectrophysiological recordings were performed in freshly isolated rabbit coronary arterial smooth muscle cells.Key findingsThe Kv current amplitude was inhibited by LY294002 in a dose-dependent manner, with a K_d value of 1.48 μM. Without alteration of the kinetics of activation, LY294002 accelerated the decay rate of Kv channel inactivation. The rate constants of association and dissociation for LY294002 were 1.83 ± 0.01 μM^? 1 s^? 1 and 2.59 ± 0.14 s^? 1, respectively. Application of LY294002 had no significant impact on the steady-state activation or inactivation curves. In the presence of LY294002, the recovery time constant from inactivation was increased, and Kv channel inhibition increased under train pulses (1 or 2 Hz). This indicates that LY294002-induced Kv channel inhibition is use-dependent. Furthermore, pretreatment with another PI3K inhibitor, wortmannin (10 μM), did not affect the Kv current, and did not change the inhibitory effect of LY294002.SignificanceBased on these results, we suggest that LY294002 directly blocks Kv current irrespective of PI3K inhibition.     

Evaluación de la utilización de las prestaciones específicas de los sistemas de infusión subcutánea de insulina y su relación con el control metabólico en pacientes con diabetes tipo 1

Background and objectivePatients with type 1 diabetes (T1DM) treated with continuous subcutaneous insulin infusion (CSII) have available several specific features of these devices. The aim of this study was to evaluate the relationship between real use of them and the degree of glycemic control in patients using this therapy.Patients and methodsForty-four T1DM patients on CSII therapy with or without real-time continuous glucose monitoring (CGM) were included. Data from 14 consecutive days were retrospectively collected using the therapy management software CareLink Personal/Pro^® and HbA1c measurement performed at that period. The relationship between the frequency of usie of specific features of insulin pumps (non-sensor augmented or sensor-augmented) and glycemic control was analyzed.ResultsMean HbA1c in the group was 7.5 ± .8%. Mean daily number of boluses administered was 5.1 ± 1.8, with 75.4% of them being bolus wizards (BW). Daily number of boluses was significantly greater in patients with HbA1c < 7.5% than in those with HbA1c > 7.5% (5.3 ± 1.6 vs. 4.3 ± 1.6, P = .056). There was a trend to greater use of BW in patients with better control (82.8 ± 21.4% vs. 69.9 ± 29.1%, P = .106). HbA1c was lower in patients using CGM (n = 8) as compared to those not using sensor-augmented pumps (7.6 ± .8 vs 7.1 ± .7, P = .067), but the difference was not statistically significant.ConclusionsMore frequent use of BW appears to be associated to better metabolic control in patients with T1DM using pump therapy. In standard clinical practice, augmentation of insulin pump with CGM may be associated to improved glycemic control.     

Limited versus total epithelial debridement ocular surface injury: Live fluorescence imaging of hemangiogenesis and lymphangiogenesis in Prox1-GFP/Flk1::Myr-mCherry mice

BackgroundImmunohistochemical staining experiments have shown that both hemangiogenesis and lymphangiogenesis occur following severe corneal and conjunctival injury and that the neovascularization of the cornea often has severe visual consequences. To better understand how hemangiogenesis and lymphangiogenesis are induced by different degrees of ocular injury, we investigated patterns of injury-induced corneal neovascularization in live Prox1-GFP/Flk1::myr-mCherry mice, in which blood and lymphatic vessels can be imaged simultaneously in vivo.MethodsThe eyes of Prox1-GFP/Flk1::myr-mCherry mice were injured according to four models based on epithelial debridement of the: A) central cornea (a 1.5-mm-diameter circle of tissue over the corneal apex), B) total cornea, C) bulbar conjunctiva, and D) cornea + bulbar conjunctiva. Corneal blood and lymphatic vessels were imaged on days 0, 3, 7, and 10 post-injury, and the percentages of the cornea containing blood and lymphatic vessels were calculated.ResultsNeither central corneal nor bulbar conjunctival debridement resulted in significant vessel growth in the mouse cornea, whereas total corneal and corneal + bulbar conjunctival debridement did. On day 10 in the central cornea, total cornea, bulbar conjunctiva, and corneal + bulbar conjunctival epithelial debridement models, the percentage of the corneal surface that was occupied by blood vessels (hemangiogenesis) was 1.9 ± 0.8%, 7.14 ± 2.4%, 2.29 ± 1%, and 15.05 ± 2.14%, respectively, and the percentage of the corneal surface that was occupied by lymphatic vessels (lymphangiogenesis) was 2.45 ± 1.51%, 4.85 ± 0.95%, 2.95 ± 1.27%, and 4.15 ± 3.85%, respectively.ConclusionsSubstantial corneal debridement was required to induce corneal neovascularization in the mouse cornea, and the corneal epithelium may therefore be partially responsible for maintaining corneal avascularity.General significanceOur study demonstrates that GFP/Flk1::myr-mCherry mice are a useful model for studying coordinated hemangiogenic and lymphangiogenic responses.     

Muscular activation patterns during active prone hip extension exercises

BackgroundChanges in activation patterns of hip extensors and pelvic stabilizing muscles are recognized as factors that cause low back disorders and these disturbances could have an impact on the physiological loading and alter the direction and magnitude of joint reaction forces.ObjectiveTo investigate activation patterns of the gluteus maximus, semitendinosus and erector spinae muscles with healthy young individuals during four different modalities of therapeutic exercise.MethodsThirty-one volunteers were selected: (16 men and 15 women), age (24.5 ± 3.47 years), body mass of 66.89 ± 11.89 kg and a height of 1.70 ± 0.09 m). They performed four modalities of therapeutic exercise while the electromyographic activity of the investigated muscles was recorded to determine muscle pattern activation for each exercise.ResultsRepeated measure ANOVA revealed that muscle activation patterns were similar for the four analyzed exercises, starting with the semitendinosus, followed by the erector spinae, and then, the gluteus maximus. The gluteus maximus was the last activated muscle during hip extension associated with knee flexion (p < 0.0001), knee extension (p < 0.0001), and with lateral rotation and knee flexion (p < 0.05).ConclusionFindings of the present study suggested that despite individual variability, the muscle firing order was similar for the four therapeutic exercises.     

Formulation of Pseudomonas chlororaphis strains for improved shelf life

Formulations of Pseudomonas strains with long-term shelf life are needed for commercial use in biological disease control and growth promotion in crops. In the present work Pseudomonas chlororaphis (Pc) 63-28 formulated with coconut fiber [moisture content (MC) of 80%], talc (MC 8%) or peat (MC 40%), with or without the addition of carboxymethylcellulose or xanthan gum, and formulations of Pc 63-28 and P. chlororaphis TX-1 in coconut fiber with water contents (v:v) of 75%, 45%, and 25%, were evaluated in terms of shelf life and cell viability. The shelf life of Pc 63-28 was longer when formulated in coconut fibre with a MC was 80% than in the other formulations and longer at 3 ± 1 °C compared to 22 ± 1 °C. Densities of viable Pc 63-28 cells in coconut fiber stored at 3 ± 1 °C did not decline significantly during 224 days when the MC was 80% and within 120 days at 75% MC. Densities of Pc TX-1 in coconut fiber of 75% MC did not decline within 60 days at 3 ± 1 °C. P. chlororaphis 63-28 survived longer in deionized water and buffer than in canola oil. Cells of Pc 63-28 cells formulated in coconut fibre of 80% MC after storage for 140 days at 3 ± 1 °C in coconut fiber improved hydroponic growth of hydroponic lettuce and better than cells freshly recovered from culture. We conclude that coconut fiber is a carrier of superior performance in maintaining shelf life of Pseudomonas strains. The observed shelf life would be sufficient for practical use of Pseudomonas strains as tools for disease control and growth promotion in crops.     

The influence of pre-operative risk on the number of circulating endothelial progenitor cells during cardiopulmonary bypass

Background aimsThe number of circulating endothelial progenitor cells (EPC) depends on cytokine release and is also associated with cardiovascular risk factors. During cardiopulmonary bypass (CPB) the endothelium is the first organ to be affected by mechanical and immunologic stimuli. We hypothesized that the magnitude of EPC mobilization by CPB correlates with the pre-operative cardiovascular morbidity profile.MethodsEPC were quantified in blood samples from 30 patients who underwent cardiac surgery by magnetic bead isolation and fluorescence-activated cell sorting (FACS) analysis, based on concomitant expression of CD34, CD133 and CD309. Patients were divided into two groups based on the European System for Cardiac Operative Risk Evaluation (EuroSCORE): low risk (LR) and high risk (HR). Ten healthy volunteers served as controls. Samples were obtained before the start of CPB and at 1 and 24 h post-operatively. Plasma samples were collected for determination of release levels of cytokines and growth factors.ResultsAll CPB patients showed a significantly reduced basal number of EPC compared with healthy individuals (LR 5.60 ± 0.39/mL, HR 3.89 ± 0.34/ mL, versus control 0.807 ± 0.82/mL, P = 0.012 versus LR, P < 0.001 versus HR). CPB induced EPC release that peaked 1 h after surgery (pre-operative 4.79 ± 0.32/mL, 1 h 57.49 ± 5.31/mL, 24 h 6.67 ± 1.05/mL, P < 0.001 pre-operative versus 1 h, P < 0.001 pre-operative versus 24 h) and was associated with the duration of CPB. However, EPC release was significantly attenuated in HR patients (33.09 ± 3.58/mL versus 81.89 ± 4.36/mL at 1 h after CPB, P < 0.0001) and inversely correlated with the pre-operative EuroSCORE. Serum granulocyte–colony-stimulating factor (G-CSF), stem cell factor (SCF) and vascular endothelial growth factor (VEGF) levels increased throughout the observation period and were also correlated with the EPC count.ConclusionsCardiovascular risk factors influence the mobilization of EPC from the bone marrow after stimulation by CPB. This could be secondary to impaired mobilization or the result of increased EPC turnover, and may have implications for future cell therapy strategies in cardiac surgical patients.     

Relaxation by β3-adrenoceptor agonists of the isolated human internal anal sphincter

AimsIn this study, responses of β₃-adrenoceptor agonists were examined on human isolated internal anal sphincter (IAS) in order to explore their relaxant effects on hypertonicity of IAS.Main methodsThe relaxant efficacy (E_max) and potency (? logIC₅₀) of BRL37344 and SR58611A, β₃-adrenoceptor agonists, were examined in contracted IAS muscle strips. The presence of β₃-adrenoceptors, and changes in intracellular calcium and cyclic nucleotide levels in IAS muscle were tested by Western blotting, epifluorescence microscopy and enzyme immunoassay, respectively.Key findingsBRL37344 and SR58611A relaxed contracted IAS muscle (E_max = 27 ± 3% and 35 ± 3%; -logIC₅₀ = 6.26 ± 0.24 and 4.87 ± 0.13; respectively). These relaxant responses were blocked by SR59230A, a selective β₃-antagonist but not by β₁/β₂-selective antagonists, neuronal inhibitor or inhibition of nitric oxide synthase. The E_max of β₃-agonists was similar to that of β₂-selective agonists but smaller than that of isoprenaline (nonselective agonist) or β₁-selective agonists. BRL37344 (100 μM) increased cAMP (1.5-fold) without cGMP change, and depressed intracellular calcium signal. β₃-Adrenoceptor expression was smaller than that of β₁- and β₂-adrenoceptors.SignificanceThis is the first study demonstrating the presence of β₃-adrenoceptor in human IAS muscle and β₃-mediated relaxation of augmented sphincter tone. However, direct β₃-relaxation appears smaller than that obtained for nonselective agonists which may limit their potential use in the treatment of anorectal hypertonicity disorders.     

Semi-automated program for analysis of local Ca2+ spark release with application for classification of heart cell type

Local Ca²⁺ spark releases are essential to the Ca²⁺ cycling process. Thus, they play an important role in ventricular and atrial cell contraction, as well as in sinoatrial cell automaticity. Characterizing their properties in healthy cells from different regions in the heart can reveal the basic biophysical differences among these regions. We designed a semi-automatic Matlab Graphical User Interface (called Sparkalyzer) to characterize parameters of Ca²⁺ spark release from any major cardiac tissue, as recorded in line-scan mode with a confocal laser-scanning microscope. We validated the algorithm on experimental images from rabbit sinoatrial, atrial, and ventricular cells loaded with Fluo-4 AM. The program characterizes general image parameters of Ca²⁺ transients and sparks: spark duration, which indicates for how long the spark provides Ca²⁺ to the closed intracellular mechanisms (typical value: 25 ± 1, 23 ± 1, 26 ± 1 ms for sinoatrial, atrial, and ventricular cells, respectively); spark amplitude, which indicates the amount of Ca²⁺ released by a single spark (1.6 ± 0.1, 1.6 ± 0.2, 1.4 ± 0.1 F/F₀ for sinoatrial, atrial, and ventricular cells, respectively); spark length, which is the length of the Ca²⁺ wavelets fired out of a row of ryanodine receptors (5 ± 0.1, 5 ± 0.2, 3.4 ± 0.3 μm for sinoatrial, atrial, or ventricular cells, respectively) and number of sparks (0.14 ± 0.02, 0.025 ± 0.01, 0.02 ± 0.01 for 1 μm in 1 s for sinoatrial, atrial, and ventricular cells, respectively). This method is reliable for Ca²⁺ spark analysis of sinoatrial, atrial, or ventricular cells. Moreover, by examining the average value of Ca²⁺ spark characteristics and their scattering around the mean, atrial, ventricular and sinoatrial cells can be differentiated.     

Protective effects of losartan in mice with chronic viral myocarditis induced by coxsackievirus B3

AimTo investigate whether losartan has protective effects in mice with chronic viral myocarditis induced by coxsackievirus B3 (CVB3).Main methodsThirty two male Balb/c mice were intraperitoneally injected with CVB3 (10 × TCID₅₀) to induce chronic viral myocarditis (CVM). Losartan at 12.5 mg/kg (n = 16) or normal saline (n = 16) were orally administered daily for 28 days to these mice. Uninfected mice (n = 6) were used as controls. On day 29, all mice underwent anesthesia and echocardiography prior to sacrifice. Serum IL-17, IL-4, IFN-γ and TNF-α levels were measured by enzyme-linked immunosorbent assay, and cardiac tissues were histologically examined after hematoxylin & eosin staining. In addition, the effect of losartan on the virus titers in primary cultured neonatal rat cardiomyocytes infected with CVB3 was measured on Hep-2 cells at 72 h post infection.Key findingsMice infected with CBV3 had significantly increased mortality, heart/body weight ratios, necrosis and inflammatory scores and decreased cardiac ejection fractions, compared with the controls (all P < 0.05). Losartan significantly decreased mortality from 40.0% to 12.5%, heart/body weight ratios from 7.08 ± 2.17 to 4.15 ± 0.99, and necrosis and inflammatory scores from 3.33 ± 0.50 to 2.50 ± 0.65 (all P < 0.05), and increased ejection fractions from 55.80 ± 9.25 to 72.31 ± 12.15 (P < 0.05). Losartan significantly enhanced IL-4, and decreased IFN-γ, TNF-α and IL-17 (all P < 0.05). In the in vitro experiment, losartan had no influence on virus titers.SignificanceLosartan protects mice against CVB3-induced CVM, most likely through upregulating Th2 responses, and down-regulating Th1 and Th17 responses.     

Are chronic neck pain,scapular dyskinesis and altered scapulothoracic muscle activity interrelated?: A case-control study with surface and fine-wire EMG

ObjectivesThe function of the scapula is important in normal neck function and might be disturbed in patients with neck pain. The surrounding muscular system is important for the function of the scapula. To date, it is not clear if patients with idiopathic neck pain show altered activity of these scapulothoracic muscles. Therefore, the objective of this study was to investigate differences in deeper and superficial lying scapulothoracic muscle activity between patients with idiopathic neck pain and healthy controls during arm elevation, and to identify the influence of scapular dyskinesis on muscle activity.MethodsScapular dyskinesis was rated with the yes/no method. The deeper lying (Levator Scapulae, Pectoralis Minor (Pm) and Rhomboid major) and superficial lying (Trapezius and Serratus Anterior) scapulothoracic muscles’ activity was investigated with fine-wire and surface EMG, respectively, in 19 female subjects with idiopathic neck pain (age 28.3 ± 10.1 years, average duration of neck pain 45.6 ± 36.3 months) and 19 female healthy control subjects (age 29.3 ± 11.7 years) while performing scaption and towel wall slide. Possible interactions or differences between subject groups, scapular dyskinesis groups or phases of the task were studied with a linear mixed model.ResultsHigher Pm activity during the towel wallslide (p = 0.024, mean difference 8.8 ± 3.3% MVIC) was shown in patients with idiopathic neck pain in comparison with healthy controls. For the MT, a significant group 1 dyskinesis interaction effect was found during scaption which revealed that patients with neck pain and scapular dyskinesis showed lower Middle Trapezius (MT) activity in comparison with healthy controls with scapular dyskinesis (p = 0.029, mean difference 5.1 ± 2.2% MVIC).ConclusionsIn the presence of idiopathic neck pain, higher Pm activity during the towel wallslide was found. Patients with neck pain and scapular dyskinesis showed lower MT activity in comparison with healthy controls with scapular dyskinesis during scaption. Scapular dyskinesis did not have a significant influence on scapulothoracic muscle activity.     

Radiological properties of 3D printed materials in kilovoltage and megavoltage photon beams

PurposeThis study evaluates the radiological properties of different 3D printing materials for a range of photon energies, including kV and MV CT imaging and MV radiotherapy beams.MethodsThe CT values of a number of materials were measured on an Aquilion One CT scanner at 80 kVp, 120 kVp and a Tomotherapy Hi Art MVCT imaging beam. Attenuation of the materials in a 6 MV radiotherapy beam was investigated.ResultsPlastic filaments printed with various infill densities have CT values of −743 ± 4, −580 ± 1 and −113 ± 3 in 120 kVp CT images which approximate the CT values of low-density lung, high-density lung and soft tissue respectively. Metal-infused plastic filaments printed with a 90% infill density have CT values of 658 ± 1 and 739 ± 6 in MVCT images which approximate the attenuation of cortical bone. The effective relative electron density RED_eff is used to describe the attenuation of a megavoltage treatment beam, taking into account effects relating to the atomic number and mass density of the material. Plastic filaments printed with a 90% infill density have RED_eff values of 1.02 ± 0.03 and 0.94 ± 0.02 which approximate the relative electron density RED of soft tissue. Printed resins have RED_eff values of 1.11 ± 0.03 and 1.09 ± 0.03 which approximate the RED of bone mineral.Conclusions3D printers can model a variety of body tissues which can be used to create phantoms useful for both imaging and dosimetric studies.