Trial of roxithromycin in subjects with asthma and serological evidence of infection with Chlamydia pneumoniae.

An association has been reported between chronic infection with Chlamydia pneumoniae and the severity of asthma, and uncontrolled observations have suggested that treatment with antibiotics active against C. pneumoniae leads to an improvement in asthma control. We studied the effect of roxithromycin in subjects with asthma and immunoglobulin G (IgG) antibodies to C. pneumoniae > or = 1:64 and/or IgA antibodies > or = 1:16. A total of 232 subjects, from Australia, New Zealand, Italy, or Argentina, were randomized to 6 wk of treatment with roxithromycin 150 mg twice a day or placebo. At the end of 6 wk, the increase from baseline in evening peak expiratory flow (PEF) was 15 L/min with roxithromycin and 3 L/min with placebo (p = 0.02). With morning PEF, the increase was 14 L/min with roxithromycin and 8 L/min with placebo (NS). In the Australasian population, the increase in morning PEF was 18 L/min and 4 L/min, respectively (p = 0.04). At 3 mo and 6 mo after the end of treatment, differences between the two groups were smaller and not significant. Six weeks of treatment with roxithromycin led to improvements in asthma control but the benefit was not sustained. Further studies are necessary to determine whether the lack of sustained benefit is due to failure to eradicate C. pneumoniae.

Effect of caffeine and high potassium on normal and dystrophic mouse EDL muscles at various developmental stages.

EDL muscles from normal and dystrophic (dy2j) mice of various ages were examined. Muscles were divided into three groups according to age: 7 to 14 days postnatal, 16 to 21 days postnatal, and 6 months old, to assess age and/or phenotype related differences in the muscle response to caffeine or high K+. The response of normal muscles to caffeine decreased with age and reached adult characteristics between the second and third week of postnatal life. Their response to high K+ also changed during postnatal development; specifically, the time taken to recover to 50% pretest twitch tension decreased with age, probably reflecting developmental changes in Cl- conductance. Up to 21 days of age, the sensitivity of dystrophic muscles to both caffeine and high K+ was essentially similar to normal, while marked differences were observed in the adult. Taken altogether, our results suggest that while the maturation of a number of systems might be delayed in dystrophic muscles at preclinical stages of the disease, their e-c coupling and SR function (Ca2+ release and reuptake) appear to be quite normal. Our results further suggest that the "abnormal" responses of dystrophic muscles at more advanced stages of the disease, when challenged by drugs acting on either of these systems, may be explained in terms of changes in muscle fiber type proportions.

Effect of low Ca2+ solution on muscle contraction of developing, preclinical dystrophic (dy2j) mice.

EDL muscles from normal and dystrophic (dy2j) mice aged 7 to 21 days of postnatal life were examined. Muscles were divided into 2 groups according to age, 7 to 14 days and 16 to 21 days postnatal, so as to assess age- and/or phenotype-related differences in the muscle response to low Ca2+ solution. Tension production was already much impaired in "predystrophic" muscles. At this stage, however, there was essentially no difference in twitch kinetics between normal and dystrophic muscles. Upon exposure to low Ca2+ solution, twitch responses of both normal and dystrophic muscles declined in a similar manner. In the youngest animals studied (7 to 14 days), the tetanic responses of both normal and dystrophic muscles to low Ca2+ solution were also similar. In animals 15 to 21 days old, however, the tetanic tension developed in low Ca2+ solution by dystrophic muscles, was significantly less than that of normal. Moreover, under these conditions (i.e., in low Ca2+ solution), and following tetanic stimulation, the membrane potential of dystrophic muscles in this age group was significantly more depolarized than that of normal muscles. Our results suggest that the ability of the cell to deal with extracellular Ca2+ is normal in predystrophic muscles up to 21 days of postnatal life. The results also clearly point to the fragility of the membrane in these muscles.

Effect of low extracellular calcium and ryanodine on muscle contraction of the mouse during postnatal development.

We have examined the effects of low Ca2+ solutions, Co2+, and ryanodine on the isometric tension and contraction speed of isolated, developing mouse EDL muscles. Twitch responses of young muscles (7-14 days postnatal) were more sensitive to lowered [Ca2+]o than those of more fully developed muscles (22-35 days postnatal). Responses of EDL muscles from a middle-aged group (15-21 days postnatal) were intermediate between the two other groups. Overall, the time course of contraction in a single twitch was accelerated by low [Ca2+]o. Ca(2+)-free solution induced a 7.95 and 9.25 mV depolarization in young and "old" muscle fibres, respectively. The presence of cobalt ions (5 mM) in the Krebs solution had a similar effect as Ca(2+)-free Krebs in terms of reduction of the isometric twitch and tetanic tensions of EDL muscles from the various age groups. In contrast, the shortening of the contraction time seen with Ca(2+)-free solution did not take place following exposure to Co(2+)-containing solutions. Finally, young (7-14 days postnatal) muscles were less sensitive to the inhibitory action of ryanodine on the twitch compared with more fully developed muscles (22-35 days postnatal). Taken together, our results indicate that from birth to maturity, there is a gradual change in the spectrum of calcium utilization for the contractile process.

Response of a fast muscle from normal and dystrophic (dy2j) mice to a local decrease in extracellular Ca2+ induced at different stages of postnatal life.

It has previously been reported that reducing Ca2+ entry into muscle fibres was beneficial to dystrophic muscles. In this study, we examined the effect, on the force output and contractile properties of the tibialis anterior muscle, of a local decrease in extracellular Ca2+, produced in normal and dystrophic mice at various stages of postnatal life by applying a small strip of silicon rubber containing a calcium chelator (BAPTA). Lowering extracellular Ca2+ in this way at an early stage of postnatal life (11-16 days) interferes with normal development in that, 3-5 weeks after the initial operation, the treated TA muscles from normal mice are weaker and their contractile speed is slower than that of their untreated counterparts. In contrast, the same procedure has a beneficial effect on dystrophic muscles in that they produce more force than untreated controls. Our results show that these changes are not related to changes in the total number of muscle fibres or fibre type proportions. These changes are temporary and by 8-12 weeks after the operation, the treated muscles are indistinguishable from controls. Finally, our results also indicate that skeletal muscles from older animals, both normal and dystrophic, become insensitive to this manipulation. These results provide the first evidence for a difference in the sensitivity of normal immature and normal adult skeletal muscles to their extracellular Ca2+ environment. They also suggest that in this context, dystrophic muscles might already differ from normal at a stage prior to the clinical expression of the symptoms of the disease.

Long term effect of low frequency chronic electrical stimulation on the fast hind limb muscles of dystrophic mice.

Low frequency chronic electrical stimulation can have a beneficial effect on dystrophic muscles. The present study was undertaken to assess the long term effect of such stimulation on the fast hind limb muscles of dystrophic mice. The relationship between the changes induced by stimulation and the initial condition of the dystrophic muscles, as well as other factors which might contribute to this relationship, were examined. The stimulation induced an increase in the force output of weak dystrophic muscles and a speeding of their time course of contraction and relaxation, as well as an increase in their fatigue resistance. In relatively strong dystrophic muscles, the stimulation induced similar changes in contractile speed and fatigue characteristics, but it led to a slight decrease in force output. Our results suggest that the stimulation promotes the growth and differentiation of the small regenerating fibres known to be present in the diseased muscles and, in addition, induces an increase in the mitochondrial content of the muscle fibres. Our results indicate that these effects are not permanent.

Response of dystrophic muscles to reduced load.

We have previously reported that, in dystrophic mice, functional overload has a damaging effect on the tibialis anterior (TA) muscle. In the present study, we have examined the effect of a load reduction on the TA and extensor digitorum longus (EDL) muscles. Our results show that reducing the passive load to which these muscles are subjected in dystrophic mice by resecting the Achilles tendon has a beneficial effect. The force output of the "released" EDL muscle improved, while the time course of contraction and relaxation of the "released" TA muscle became faster. Also in this muscle, resistance to fatigue became significantly greater. Low frequency electrical stimulation of the "released" muscles via implanted electrodes had little effect on their force output. It led, however, to a relative speeding of their time course of contraction and relaxation and to a further increase in their resistance to fatigue. Taken together, our results suggest that the beneficial effect of low frequency electrical stimulation on the force output of weak dystrophic muscles, described in the preceding paper, might be conditioned by the load to which these muscles are subjected.

Developmental changes in succinate dehydrogenase activity in muscle fibers from normal and dystrophic mice.

The question of whether or not the development of dystrophic muscles is similar to that of normal muscles, prior to the manifestations of the symptoms of the disease, is investigated here. The developmental change in the activity of succinate dehydrogenase was therefore measured in individual fibers of prospectively dystrophic muscles from 10- to 28-day-old mice (strain C57Bl/6J dy2j) and compared with that of muscles from normal mice of the same age. It was found that up to 10 days of age, muscle fibers from normal and prospective dystrophic animals had low succinate dehydrogenase activities, and were all more or less uniform. Thereafter in the normal muscle the overall activity of the enzyme increased and the fibers became more heterogeneous with age. By 21 days the extensor digitorum longus muscle resembled that of the adult. At that time, fibers from prospectively dystrophic muscles had lower succinate dehydrogenase activities and were more homogeneous. Thus fibers from prospectively dystrophic muscles fail to achieve their adult characteristics by 21 days. On the basis of these results, it is suggested that muscle maturation is retarded in dystrophic animals.

Response of normal and dystrophic muscles to increased functional demand.

Immature fast muscles appear to be unfavorably influenced by excessive activity and there is evidence suggesting that the maturation of muscles from animal models of muscular dystrophy and patients suffering from Duchenne dystrophy is impaired. We therefore examined the effects of chronic functional overload applied at different stages of postnatal life on a fast muscle in normal and dystrophic mice (C57B1/6J dy2j/dy2j). "Overload" of tibialis anterior muscle was produced by removal of its synergist extensor digitorum longus in one hind limb. Our results suggest that increased functional demand can be damaging to immature muscles and that in dystrophic animals, the inability to adjust to overload persists into adult life.

Muscle development in mdx mutant mice.

Mechanical and contractile properties of tibialis anterior (TA) muscles from X-linked muscular dystrophic (mdx) mutant mice at different stages of development are compared to those of muscles from normal control animals. There is no difference between the tension output, speeds of contraction and relaxation, and weight of TA muscles from mutant adults and normal control animals. However, it is found that in 3-4-week-old mutant animals, tension output and muscle weight are very much reduced, and half relaxation time is prolonged. Thus, during this stage of development, muscles from mdx mice do not function properly. Histological examination of these muscles provides further evidence that, in these animals, rapid muscle destruction occurs at a particular time of development and that it is followed by complete recovery. This new mutant therefore presents an interesting case of muscle destruction and rapid regeneration. However, it is not an adequate model for Duchenne muscular dystrophy.

Elimination of polyneuronal innervation in a fast muscle of normal and dystrophic mice.

The changes in the pattern of innervation of extensor digitorum longus (e.d.l.) during post-natal development was studied in normal and dystrophic mice. As in other mammals, individual muscle fibres of new-born mice are supplied by more than one axon. Up to 10 days after birth there was no difference in the extent of this polyneuronal innervation between normal and dystrophic muscle fibres. During post-natal development the polyneuronal innervation gradually disappeared. In normal e.d.l. muscles the rate of the elimination of polyneuronal innervation was faster during the first 10 post-natal days and then slowed down. By 16 days the final value of less than 10% of muscle fibres receiving more than one input was reached. In the dystrophic muscles the rate of elimination was similar to normal up to 10 days of age, but continued to decrease rapidly so that already by 11 days of age polyneuronal innervation was reduced to its final level of less than 10%. Thus the elimination of polyneuronal innervation was completed at least 3 days earlier in the dystrophic animals. It is suggested that the increased nerve activity said to be present in dystrophic mice could account for this finding.

Effect of chronic electrical stimulation at low frequency on the passive membrane properties of muscle fibers from dystrophic mice.

It has been reported that chronic electrical stimulation at low frequency applied to dystrophic muscles has a beneficial effect. In this study, the effect of this treatment on the passive membrane properties of muscle fibers from dystrophic mice was followed. Cable properties were assessed by the two-microelectrodes DC method and spacial decay analysis. Earlier results showing a decrease in resting potential, an increase in input resistance and in specific membrane resistance in muscle fibers from dystrophic mice were confirmed. In addition, the specific membrane capacitance of these muscle fibers was found to be lower than normal. This suggests that the membrane properties of fibers from dystrophic muscles are similar to those of immature muscle fibers. Muscle fibers from dystrophic animals that were stimulated for 2 to 4 weeks had membrane properties similar to those from normal muscles. This indicates that electrical stimulation at low frequency for 2 to 4 weeks restores membrane properties of dystrophic muscle fibers to normal and we suggest that an appropriate pattern of stimulation induces the maturation of dystrophic muscle fibers.