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.
We examined the effect of running training on age-related changes in cardiac myosin isozyme composition in rats. Female Fischer 344 rats (6, 12, 20, and 27 months old) were divided into two groups: ...sedentary control and trained. The trained group rats were trained by treadmill running for up to 60 minutes per day, 5 days per week for 8 weeks at up to 30 m per minute. In sedentary control rats, the proportion of V1 myosin, that is, alpha-myosin heavy chain (MyHC) isoform, decreased progressively from 6 to 27 months of age. In the younger age groups (6 or 12 months old), there was a shift from V1 myosin to V3 myosin (beta-MyHC isoform) in trained hearts. However, the training program did not induce a cardiac myosin isozyme transition in older rats (20 or 27 months old). These results suggest that the mechanisms mediating the responses of cardiac muscle to running training alter during aging.
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. The effects of the nerve and of thyroid hormone on these developing fast muscles therefore appear synergistic. In the adult AT and EDL, hypothyroidism causes a significant rise in proportions of slow MHC, which selectively accumulates in type IIa and not IIb fibers. This pattern of accumulation is not a simple recapitulation of early programs of slow MHC expression.
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.
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.
We examined the effects of prolonged voluntary wheel-running on skeletal muscle functional and/or structural characteristics in rats. Male Sprague-Dawley rats (5 weeks old) were divided into five ...groups: (1) 15W-SC, sedentary controls housed in normal plastic cages until age 15 weeks; (2) 15W-VE, housed in a voluntary-exercise (running-wheel) device equipped with housing space until age 15 weeks; (3) 35W-SC, housed in normal plastic cages until age 35 weeks; (4) 35W-VE, housed in the voluntary-exercise device until age 35 weeks, and (5) 35W-MVE, housed in normal plastic cages until age 15 weeks, then in the voluntary-exercise device from age 16 weeks to 35 weeks ("middle age"). At the end of each rat's experimental period, the plantaris muscle was dissected from each hindlimb for analysis of the muscle's functional and/or structural characteristics. Total running distance was similar in 15W-VE and 35W-VE, both being significantly greater than in 35-MVE. The percentage of type IIb myosin heavy chain isoform was significantly lower in each VE group than in the corresponding SC group. This shift from type IIb was significantly greater for 35W-VE than for the other VE groups, which were similar to each other. The cross-sectional area of type IIx fibers was significantly greater in 35W-VE than in 35W-SC, but this was not true for 15W-VE versus 15W-SC or for 35W-MVE versus 35W-SC. No significant difference in citrate synthase activity was detected between any VE group and the corresponding SC group. These results suggest that a prolongation of voluntary wheel-running leads to some advantageous enhancements of functional and/or structural characteristics in rat plantaris.
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.
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 (17131) 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 FDl 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.
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