Therapeutic vaccination based on side population cells transduced by the granulocyte-macrophage colony-stimulating factor gene elicits potent antitumor immunity.

Among cancer immunotherapies, granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transduced tumor cell vaccine (GVAX) therapies appear promising and have been shown to be safe and effective in multiple clinical trials. However, the antitumor efficacies of GVAX therapy alone are in some cases limited. Here we showed that GVAX therapy targeting cancer stem cells (CSCs) substantially suppressed tumor development in syngeneic immunocompetent mice recapitulating normal immune systems. CSCs were isolated as side population (SP) cells from 4T1 murine breast carcinoma cell line and transduced with GM-CSF gene delivered by non-transmissible Sendai virus (4T1-SP/GM). Impaired tumorigenicity of subcutaneously injected 4T1-SP/GM depended on CD8+ T cells in concert with CD4+ T cells and natural killer cells. Mice therapeutically vaccinated with irradiated 4T1-SP/GM cells had markedly suppressed tumor development of subcutaneously transplanted 4T1-SP cells compared with those treated with irradiated cells of non-transduced 4T1-SP cells or non-SP (4T1-NSP/GM) cells. Tumor suppression was accompanied by the robust accumulation of mature dendritic cells at vaccination sites and T-helper type 1-skewed systemic cellular immunity. Our results suggested that CSC cell-based GVAX immunotherapy might be clinically useful for inducing potent tumor-specific antitumor immunity.

Effects of joint immobilization on firing rate modulation of human motor units.

We investigated the effects of 6 weeks of immobilization on firing rate modulation in motor units in the first dorsal interosseous (FDI) of human volunteers. The middle finger, index finger and thumb were immobilized for a period of 6 weeks in a fibre-glass cast, which kept FDI in a shortened position. During isometric contraction at 20, 40, 60 and 80 % of maximal voluntary contraction (MVC) (index finger abduction), motor unit action potentials were recorded from the FDI using a tungsten microelectrode, and the relationship between voluntary force and mean firing rate (MFR) was obtained by plotting the MFR of each motor unit action potential train as a function of voluntary force. Four recording sessions were held for each subject: before immobilization, after 3 and 6 weeks of immobilization, and after a 6 week recovery period. As a result of immobilization, FDI volume (as measured by computerized tomography (CT) scanning) decreased, with an accompanying reduction in aggregate EMG activity per day (P < 0.01). The force measured during MVC also decreased (P < 0.05). The slope of the relationship between voluntary force and MFR was significantly decreased after immobilization, as was the range of firing rate modulation (P < 0.01). Maximal MFR, estimated from the relationship between voluntary force and MFR, was decreased (P < 0.05). MFR was also plotted against voluntary force without being normalized with respect to MVC, and the slope of the regression line was decreased (P < 0.05). Voluntary force when the MFR was 15 Hz was estimated from regression equations for the absolute force-MFR relationship, and it was increased after immobilization (P < 0.05). These results suggest that firing rate modulation shows two different adaptations to joint immobilization: a restriction of motoneurone firing to the lower rates and an enhancement of the voluntary force exerted when the MFR is relatively low.

Alterations in contractile properties of human skeletal muscle induced by joint immobilization.

The effects of joint immobilization on the contractile properties of human skeletal muscle were examined using the first dorsal interosseous (FDI) muscle. The middle finger, index finger and thumb were immobilized for a period of 6 weeks, and the contractile properties of FDI were tested before immobilization, after 3 and 6 weeks of immobilization, and after a 6 week recovery period. Twitch and tetanic contractions of FDI were evoked by per-cutaneous electrical stimulation. The peak twitch tension (Pt), contraction time (CT) and half-relaxation time (1/2RT) were measured from twitch contractions, while the stimulus frequency-force relationship was obtained from the tetanic contractions (2 s) evoked using various frequencies of stimulation (10-100 Hz). The fatigability of FDI was tested using Burke's fatigue protocol.Pt was significantly increased after 6 weeks of immobilization (P < 0.05) but little alteration was observed in CT or 1/2RT. No change was noted in the FDI fatigue index throughout the immobilization period. The stimulus frequency-force relationship was shifted to the left by immobilization, indicating that a larger percentage of maximal force was evoked by the lower rates of stimulation. Indeed, the tetanic force evoked by a stimulus frequency of 10 Hz was enhanced after immobilization (P < 0.05). On the other hand, the force evoked by frequencies above 50 Hz, including maximal tetanic tension, was decreased (P < 0.05). As a result, the twitch/tetanus ratio was increased (P < 0.01) after immobilization. The changes induced by immobilization in the FDI twitch/tetanus ratio and the estimated maximal firing rate of FDI motoneurones showed a significant correlation (r = 0.80, P < 0.05). It is suggested that the changes in the contractile properties of the FDI muscle seen after joint immobilization are causally linked to the changes in firing rate modulation of FDI motoneurones.

Lack of effect of running training at two intensities on cardiac myosin isozyme composition in rats.

Little information is available regarding the influence of the intensity of endurance training over biochemical profiles in cardiac muscle. We assessed the effect of running training at two different intensities on cardiac myosin isozyme composition in rats. Male Sprague-Dawley rats (4 weeks old) were divided into four groups: sedentary control (SC), trained at 20 m/min (T20), trained at 40 m/min (T40), and weight-matched sedentary control (WMSC) groups. The T20 and T40 group rats were trained by treadmill running for 60 min/d, 5 d/week at 20 or 40 m/min, respectively, for 11 to 12 weeks. In both groups the left ventricle was significantly heavier than in WMSC animals. The ratio of left ventricle weight to body weight was significantly greater in T40 rats than in either the untrained (SC and WMSC) or trained T20 rats. Thus the extent of exercise-induced cardiac hypertrophy appears to be influenced by the intensity of running training. However, neither of the training programs (1) induced a change in cardiac myosin isozyme composition or (2) had any effect on myocardial succinate dehydrogenase or citrate synthase activity. These results suggest that although the intensity of running training may play an important role in cardiac morphological adaptation, it does not modulate the cardiac biochemical adaptation to running training.

Relation between the size of motor units and the spectral characteristics of their action potentials. kazuseki@iname.com.

OBJECTIVE: The purpose of this study was to clarify the influence of the size of an individual motor unit on the spectral characteristics of its motor unit action potentials (MUAPs). METHODS: In 4 human subjects, we first averaged the isometric force and the surface EMG signals triggered by the intramuscularly recorded action potentials of each motor unit during voluntary isometric contraction. Then, we obtained averaged twitch contraction curves, averaged MUAPs derived from surface EMG signals (S-MUAPs), and the power spectrum of the S-MUAPs. Finally, we tested for correlations among these results in each subject. RESULTS: There was a positive correlation among the mean power frequency of the S-MUAPs, the maximal amplitude of the S-MUAPs, and the maximal twitch tension of their motor unit in each subject (r = 0.58-0.85; P < 0.05). CONCLUSIONS: Since the twitch tension and the amplitude of the S-MUAPs are well-known predictors of the size of motor units, we conclude that the spectral characteristics of the S-MUAPs reflect the size of the motor unit from which they originate.

Firing rate modulation of human motor units in different muscles during isometric contraction with various forces.

To examine the factors affecting the control of human motor units, rate coding strategies of the motor units were investigated in upper limb and intrinsic hand muscles during voluntary isometric contraction of steady force levels up to 80% of maximal voluntary contraction. Numerous spike trains from single motor units were recorded from the m. first dorsal interosseous (FDI) and the m. biceps brachii (BB) of eight human subjects by means of tungsten micro-electrodes, and the mean firing rate (MFR) was calculated for each subject and inter-individual comparisons made. The MFRs of the FDI were larger than that of the BB at the higher force level, and substantial differences were not found between these muscles at the lower force level. The slope of the linear regression line of MFRs vs. exerted forces for the FDI was more than twice that for the BB. Therefore, isometric force control of the FDI depends more on the rate coding strategy. The difference in rate coding between the FDI and BB motor units may be determined by factors other than muscle fiber composition, because both muscles are known to possess a similar composition of fiber types. Possible mechanisms underlying these characteristics of rate coding strategy are considered in this report.

The mdx mouse diaphragm reproduces the degenerative changes of Duchenne muscular dystrophy.

Although murine X-linked muscular dystrophy (mdx) and Duchenne muscular dystrophy (DMD) are genetically homologous and both characterized by a complete absence of dystrophin, the limb muscles of adult mdx mice suffer neither the detectable weakness nor the progressive degeneration that are features of DMD. Here we show that the mdx mouse diaphragm exhibits a pattern of degeneration, fibrosis and severe functional deficit comparable to that of DMD limb muscle, although adult mice show no overt respiratory impairment. Progressive functional changes include reductions in strength (to 13.5% of control by two years of age), elasticity, twitch speed and fibre length. The collagen density rises to at least seven times that of control diaphragm and ten times that of mdx hind-limb muscle. By 1.5 years of age, similar but less severe histological changes emerge in the accessory muscles of respiration. On the basis of these findings, we propose that dystrophin deficiency alters the threshold for work-induced injury. Our data provide a quantitative framework for studying the pathogenesis of dystrophy and extend the application of the mdx mouse as an animal model.

Surface electromyogram spectral characterization and motor unit activity during voluntary ramp contraction in men.

The relationships were investigated between the surface electromyographic (SEMG) power spectrum analysed by the 20 order autoregressive model (AR spectrum) and underlying motor unit (MU) activity during isometric contractions increasing linearly from 0% to 80% maximal voluntary contraction. Intramuscular spikes and SEMG signals were recorded simultaneously from biceps brachii muscle; the former were analysed by a computer-aided intramuscular MU spike amplitude-frequency (ISAF) histogram and the latter subjected to AR spectral analysis. Results indicated that there was a positive correlation between the force output and the mean amplitude of the ISAF histogram but not with the mean frequency. These changes were accompanied by changes in relative power of the high frequency (100-200 Hz) peak (HL) in the AR spectrum. It was also found that there was a positive correlation between the mean amplitude of the ISAF histogram and the HL value. These data suggested that the power of the high frequency peak in the AR spectrum of the SEMG signal preferentially reflected the progressive recruitment of underlying MU according to their size. Differences between the AR spectrum and the spectrum estimated by fast Fourier transform algorithm have also been discussed.

Slow myosin in developing rat skeletal muscle.

Through S1 nuclease mapping using a specific cDNA probe, we demonstrate that the slow myosin heavy-chain (MHC) gene, characteristic of adult soleus, is expressed in bulk hind limb muscle obtained from the 18-d rat fetus. We support these results by use of a monoclonal antibody (mAb) which is highly specific to the adult slow MHC. Immunoblots of MHC peptide maps show the same peptides, uniquely recognized by this antibody in adult soleus, are also identified in 18-d fetal limb muscle. Thus synthesis of slow myosin is an early event in skeletal myogenesis and is expressed concurrently with embryonic myosin. By immunofluorescence we demonstrate that in the 16-d fetus all primary myotubes in future fast and future slow muscles homogeneously express slow as well as embryonic myosin. Fiber heterogeneity arises owing to a developmentally regulated inhibition of slow MHC accumulation as muscles are progressively assembled from successive orders of cells. Assembly involves addition of new, superficial areas of the anterior tibial muscle (AT) and extensor digitorum longus muscle (EDL) in which primary cells initially stain weakly or are unstained with the slow mAb. In the developing AT and EDL, expression of slow myosin is unstable and is progressively restricted as these muscles specialize more and more towards the fast phenotype. Slow fibers persisting in deep portions of the adult EDL and AT are interpreted as vestiges of the original muscle primordium. A comparable inhibition of slow MHC accumulation occurs in the developing soleus but involves secondary, not primary, cells. Our results show that the fate of secondary cells is flexible and is spatially determined. By RIA we show that the relative proportions of slow MHC are fivefold greater in the soleus than in the EDL or AT at birth. After neonatal denervation, concentrations of slow MHC in the soleus rapidly decline, and we hypothesize that, in this muscle, the nerve protects and amplifies initial programs of slow MHC synthesis. Conversely, the content of slow MHC rises in the neonatally denervated EDL. This suggests that as the nerve amplifies fast MHC accumulation in the developing EDL, accumulation of slow MHC is inhibited in an antithetic fashion. Studies with phenylthiouracil-induced hypothyroidism indicate that inhibition of slow MHC accumulation in the EDL and AT is not initially under thyroid regulation. At later stages, the development of thyroid function plays a role in inhibiting slow MHC accumulation in the differentiating EDL and AT.(ABSTRACT TRUNCATED AT 400 WORDS)

Change in fiber type in partially-denervated soleus muscle of the rat.

In 30% or less partially denervated muscle, the reinnervation of denervated muscle fiber may give rise to a change in motor unit size or number of muscle fibers innervated by a single motor neuron. This study was designed to evaluate changes in fiber type and contractibility of partially denervated rat soleus muscle. Partial denervation (by 30% or less) of the soleus nerve does not cause a decrease in the number of muscle fibers. A histochemical study was performed on frozen sections of the muscle. The total number of muscle fibers, atrophied fibers and type II fibers were counted. In the muscle 4 weeks after partial denervation, the number of type II fibers was fewer with a decrease of about 40% which was not significant. The twitch time to peak and half-relaxation time were not changed. The number of type II fibers was significantly decreased (p less than 0.01) after 8 weeks. There was a prolongation of contraction time. The decrease of type II fibers was extensive involving not only the denervated area but also the rest of the muscle area. The transformation of fiber type observed in partially denervated muscle may be attributed to a possible diminution of neurotrophic substances in intact motor neurons.

Population of muscle fibers during postnatal development in the rat hindlimb.

Changes in the numbers of muscle fibers during the postnatal development (1, 7, 14 and 21 days old) of rat EDL and SOL muscles were studied. Quantitative analysis of fibers was performed using cryosectioned specimens. The 1 micrometer-thick sections were taken from epon-embedded blocks of these muscle, and light micrographs were obtained. Thereafter, the same 1 micrometer-thick specimens were embedded again for EM studies. Many small cells, which could not be confirmed as myofibers at the light microscopic level, were identified as skeletal muscle fibers with certainty by electron microscopy. The population of muscle fibers apparently increased by ca. 45% in the 7-day-old rat and by ca. 67% in the 21-day-old rat, when compared with 1-day-old rat EDL and SOL muscles. This number increased gradually during postnatal development over several weeks. It was suggested that a small cell (myofiber) adjacent to a large myofiber with adherent junctions and with a common basement membrane develops into a real muscle fiber.

Effects of relative metabolic rate and heart rate variation on the performance of flight attendants.

The main work of the cabin attendants in an actual flight in service for passengers. The effects of flight attendant duties in flight differ from the effects of the same tasks performed on the ground. In this study, the relative metabolic rate (RMR) and heart rate (HR) of cabin attendants in a cruising aircraft galley and cabin are compared with those of a crew working in a mock-up apparatus on the ground. The types of work tested are: (a) oshibori (steamed towel) service, (b) soft drink service, (c) setting meal tray, (d) putting casserole on tray, (e) meal tray service, (f) walking on aisle. The RMR at each type of work during flight is as indicated: (a) 1.07-2.10, (b) 1.08-1.54, (c) 1.37-1.82, (d) 2.57-3.50, (e) 2.11-3.10 and (f) 1.84. The range of HR was: (a) 105-120, (b) 90-110, (c) 90-120, (d) 100-130 and 100-140 beats/min. In most cases, the RMR and HR levels of work done in the mock-up were lower than those recorded in flight. These results suggest that the oxygen intake of work done in flight is greater than that on a mock-up. One of the reasons might be that the cabin barometric pressure (ca. 660 torr or cabin altitude ca. 1,500 m) or an aisle inclination of about 3 degrees caused a decrease in the efficiency of oxygen intake during flight.