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Background: The rapid and massive death of cultured donor myoblasts after injection in vivo is a major problem for clinical myoblast transfer therapy (MTT). This study shows blood-borne factors are responsible and that ablating the host natural killer (NK) cell response greatly enhances the survival of such donor myoblasts., Methods: Cultured male donor myoblasts were injected into muscles of female host mice and surviving donor male DNA (myoblasts) quantified using a Y-chromosome specific (Y1) probe. Survival of donor myoblasts transfected with m144, a murine major histocompatibility complex (MHC) class I homologue that protects against NK attack, was quantified. In addition, donor myoblast survival was investigated in host mice following initial (before MTT) and sustained (repeatedly for 3 weeks after MTT) depletion of host NK1.1+ and CD4+/CD8+ cells using specific monoclonal antibodies (either alone or in combination) for up to 3 weeks after MTT, as well as in beige (deficient in NK activity) and in perforin-deficient mdx host mice., Results: A major role for blood-borne factors (especially cells) was confirmed by MTT experiments in irradiated and perfused host mice. Substantially enhanced myoblast survival was seen with donor myoblasts modified by transfection with the m144 molecule or following antibody depletion of host NK1.1+ cells and in beige host mice. Other studies support some role for CD8+ but not CD4+ cells., Conclusions: These combined data support a central role for host NK cells in the rapid initial death of donor myoblasts. The demonstrated role of NK cells provides strategies to enhance the efficacy of clinical myoblast transplantation.

Muscle wasting is a prominent feature of several systemic diseases, neurological damage and muscle disuse. The contribution of calpain proteases to muscle wasting in any instance of muscle injury or disease has remained unknown because of the inability to specifically perturb calpain activity in vivo. We have generated a transgenic mouse with muscle-specific overexpression of calpastatin, which is the endogenous inhibitor of calpains, and induced muscle atrophy by unloading hindlimb musculature for 10 days. Expression of the transgene resulted in increases in calpastatin concentration in muscle by 30- to 50-fold, and eliminated all calpain activity that was detectable on zymograms. Muscle fibres in ambulatory, transgenic mice were smaller in diameter, but more numerous, so that muscle mass did not differ between transgenic and non-transgenic mice. This is consistent with the role of the calpain-calpastatin system in muscle cell fusion that has been observed in vitro. Overexpression of calpastatin reduced muscle atrophy by 30 % during the 10 day unloading period. In addition, calpastatin overexpression completely prevented the shift in myofibrillar myosin content from slow to fast isoforms, which normally occurs in muscle unloading. These findings indicate that therapeutics directed toward regulating the calpain-calpastatin system may be beneficial in preventing muscle mass loss in muscle injury and disease.

Reduced sarcolemmal integrity in dystrophin-deficient muscles of mdx mice and Duchenne muscular dystrophy (DMD) patients has been reported to result in altered calcium homeostasis. Previous studies have shown a correlative relationship between calcium-dependent protease (calpain) activity in dystrophic muscle and muscle necrosis, but have not tested whether calpain activation precedes cell death or is a consequence of it. To test a causal relationship between calpain activation and muscle cell death in dystrophin deficiency, mdx mice were generated that overexpress a calpastatin transgene in muscle. Calpastatin (CS) is a specific, endogenous inhibitor of m- and micro -calpains that does not inhibit calpain 3 (p94). CS overexpression on a C57/BL 10 background produced no phenotype. Transgenic (Tg) mice crossed with mdx mice were tested for pathological indicators of necrosis, regeneration and membrane damage. Two lines of mice were examined, with different levels of CS overexpression. Both lines of Tg/mdx mice showed reductions in muscle necrosis at 4 weeks of age. These mice had fewer as well as smaller lesions. In addition, one line of mice had significantly less regeneration, indicating a reduction in previous necrosis. The extent of improvement correlated with the level of CS protein expression. Membrane damage, as assessed by procion orange and creatine kinase assays, was unchanged, supporting the idea that calpains act downstream of the primary muscle defect. These data suggest that calpains play an active role in necrotic processes in dystrophic muscle and that inhibition of calpains might provide a good therapeutic option for treatment of DMD.

Limb-girdle muscular dystrophy, type 2A (LGMD 2A), is an autosomal recessive disorder that causes late-onset muscle-wasting, and is due to mutations in the muscle-specific protease calpain 3 (C3). Although LGMD 2A would be a feasible candidate for gene therapy, the reported instability of C3 in vitro raised questions about the potential of obtaining a stable, high-level expression of C3 from a transgene in vivo. We have generated transgenic (Tg) mice with muscle-specific overexpression of full-length C3 or C3 isoforms, which arise from alternative splicing, to test whether stable expression of C3 transgenes could occur in vivo. Unexpectedly, we found that full-length C3 can be overexpressed at high levels in vivo, without toxicity. In addition, we found that Tg expressing C3 lacking exon 6, an isoform expressed embryonically, have muscles that resemble regenerating or developing muscle. Tg expressing C3 lacking exon 15 shared this morphology in the soleus, but not other muscles. Assays of inflammation or muscle membrane damage indicated that the Tg muscles were not degenerative, suggesting that the immature muscle resulted from a developmental block rather than degeneration and regeneration. These studies show that C3 can be expressed stably in vivo from a transgene, and indicate that alternatively spliced C3 isoforms should not be used in gene-therapy applications because they impair proper muscle development.

Many features of dystrophin-deficient muscle pathology are not clearly related to the loss of mechanical support of the muscle membrane by dystrophin. In the present review, evidence that supports a role for the immune system in promoting the pathology of dystrophinopathy is presented. The findings summarized here indicate that specific, cellular immune responses by cytotoxic T-lymphocytes and helper T-lymphocytes contribute to muscle pathology in dystrophin-deficient muscle, and that removal of specific lymphoid cell populations can reduce muscle pathology. In addition, innate immune responses may also promote dystrophinopathies by the tremendous infiltration of myeloid cell populations into the dystrophic muscle. Loss of normal redox homeostasis by dystrophin-deficient muscle may increase its sensitivity to free radical-mediated damage by myeloid cells. Collectively, the observations presented here suggest that the contribution of the immune system to dystrophinopathies may be significant, and that therapeutic approaches based upon immune interventions may ameliorate the pathological progression of dystrophin deficiency.

Duchenne muscular dystrophy (DMD) and mdx mouse dystrophy result from mutations in the dystrophin gene. Although these mutations are primarily responsible for the defects that underlie the pathology of dystrophinopathies, other factors may contribute importantly to the pathology. In the present investigation, we tested whether T cells present in mdx muscles are activated and contribute significantly to the pathological process. Flow cytometric analyses showed a significantly higher frequency of activated CD44(high) T cells in the blood and muscle of mdx mice when compared to normal B10 mice. However, the frequency of activated T cells was not elevated in mdx lymph nodes, suggesting muscle-specific T cell activation. In vivo antibody-mediated depletions of CD4(+) T cells from mdx mice significantly reduced the amount of histologically discernible muscle pathology by 61% in mdx mice, while depletion of CD8(+) T cells resulted in a 75% decrease in pathology. Finally, adoptive transfer of mdx immune cells in combination with muscle extracts resulted in muscle pathology in healthy murine recipients. These results indicate that T cells promote the mdx pathology and suggest that immune-based therapies may provide benefit to DMD patients., (Copyright 2000 Academic Press.)

We have previously demonstrated a role for T cells in Duchenne muscular dystrophy (DMD) using the mdx mouse and have shown that T cell killing of dystrophic muscle can occur through perforin-dependent and perforin-independent mechanisms. In this investigation, we explore the possibility that one perforin-independent mechanism utilized by the T cells is cytokine-based killing, specifically by tumor necrosis factor (TNF). We tested this hypothesis by generating mice that are TNF-deficient and dystrophin-deficient (TNF-/mdx). Body mass and muscle mass of the TNF-/mdx mice were significantly less than TNF+/mdx mice at 8 weeks of age. Creatine kinase levels and overall muscle strength were unchanged. Histopathology measurements showed different results in the diaphragm and quadriceps muscles. These data suggest that removal of TNF in vivo in dystrophic mice has differential effects on diaphragm and quadriceps suggesting that TNF is an unfavorable target for immunotherapy for DMD.

Previous investigations have shown that cytotoxic T lymphocytes (CTLs) contribute to muscle pathology in the dystrophin-null mutant mouse (mdx) model of Duchenne muscular dystrophy through perforin-dependent and perforin-independent mechanisms. We have assessed whether the CTL-mediated pathology includes the promotion of eosinophilia in dystrophic muscle, and thereby provides a secondary mechanism through which CTLs contribute to muscular dystrophy. Quantitative immunohistochemistry confirmed that eosinophilia is a component of the mdx dystrophy. In addition, electron microscopic observations show that eosinophils traverse the basement membrane of mdx muscle fibers and display sites of close apposition of eosinophil and muscle membranes. The close membrane apposition is characterized by impingement of eosinophilic rods of major basic protein into the muscle cell membrane. Transfer of mdx splenocytes and mdx muscle extracts to irradiated C57 mice by intraperitoneal injection resulted in muscle eosinophilia in the recipient mice. Double-mutant mice lacking dystrophin and perforin showed less eosinophilia than was displayed by mdx mice that expressed perforin. Finally, administration of prednisolone, which has been shown previously to reduce the concentration of CTLs in dystrophic muscle, produced a significant reduction in eosinophilia. These findings indicate that eosinophilia is a component of the mdx pathology that is promoted by perforin-dependent cytotoxicity of effector T cells. However, some eosinophilia of mdx muscle is independent of perforin-mediated processes.

Calpains are a ubiquitous, well-conserved family of calcium-dependent, cysteine proteases. Their function in muscle has received increased interest because of the discoveries that the activation and concentration of the ubiquitous calpains increase in the mouse model of Duchenne muscular dystrophy (DMD), but null mutations of muscle specific calpain causes limb girdle muscular dystrophy 2A (LGMD2A). These findings indicate that modulation of calpain activity contributes to muscular dystrophies by disrupting normal regulatory mechanisms influenced by calpains, rather than through a general, nonspecific increase in proteolysis. Thus, modulation of calpain activity or expression through pharmacological or molecular genetic approaches may provide therapies for some muscular dystrophies.

Mechanical stimuli can cause changes in muscle mass and structure which indicate that mechanisms exist for transducing mechanical stimuli into signals that influence gene expression. Myotendinous junctions show adaptations to modified muscle loading which suggest that these are transcriptionally distinct domains in muscle fibers that may experience local regulation of expression of structural proteins that are concentrated at these sites. Vinculin and talin are cytoskeletal proteins that are highly enriched at myotendinous junctions that we hypothesize to be subject to local transcriptional regulation. Our findings show that mechanical stimulation of muscle cells in vivo and in vitro causes an increase in the expression of vinculin and talin that is mediated by nitric oxide. Furthermore, nitric oxide-stimulated increases in vinculin and talin expression occur through a protein kinase G-dependent pathway and therefore differ from other mechanisms through which nitric oxide has been shown previously to modulate transcription. Analysis of vinculin mRNA distribution in mechanically stimulated muscle fibers shows that the mRNA is highly concentrated at myotendinous junctions, which supports the hypothesis that myotendinous junctions are distinct domains in which the expression of cytoskeletal proteins is modulated by mechanical stimuli through a nitric oxide and protein kinase G-dependent pathway.

The hypothesis that changes in muscle activation and loading regulate the expression and activity of neuronal nitric oxide (NO) synthase (nNOS) was tested using in vitro and in vivo approaches. Removal of weight bearing from rat hindlimb muscles for 10 days resulted in a significant decrease in nNOS protein and mRNA concentration in soleus muscles, which returned to control concentrations after return to weight bearing. Similarly, the concentration of nNOS in cultured myotubes increased by application of cyclic loading for 2 days. NO release from excised soleus muscles was increased significantly by a single passive stretch of 20% or by submaximal activation at 2 Hz, although the increases were not additive when both stimuli were applied simultaneously. Increased NO release resulting from passive stretch or activation was dependent on the presence of extracellular calcium. Cyclic loading of cultured myotubes also resulted in a significant increase in NO release. Together, these findings show that activity of muscle influences NO production in the short term, by regulating NOS activity, and in the long term, by regulating nNOS expression.

This study used two-day diet records to examine dietary behaviours in 504 Australian 18 year-olds in relation to gender, socio-economic status (SES) and national dietary guidelines. Fat intake exceeded 30% of energy in about 80% of subjects and was greater than 40% in about one-quarter. Saturated fat provided more than 10% of dietary energy in more than 90% of participants; less than 1% achieved a polyunsaturated to saturated fat ratio of at least one. The major food groups contributing to fat intake were convenience foods (32% in men, 28% in women) and meat (27% in men, 25% in women). Fibre intake was less than 30 g/day in 93% of women and 77% of men. Intakes of calcium, magnesium, potassium, and vitamins C and A, as a ratio of energy consumption, were greater in women than men, while sodium intake was significantly higher in men. Convenience foods were the greatest contributors to sodium intake (27% in men, 22% in women) followed by meat, bread, and soups and sauces. Greater consumption of cereals, fruit, vegetables and low-fat foods in young women of higher SES was reflected in their nutrient profile with higher intake of fibre and vitamin C and lower intake of fat. Men ate more cereals, meat and sugary foods and less fruit, vegetables and low-fat foods. Only 2.5% of men and 4.1% of women conformed with the health promotion message, widely publicised locally, to eat two fruits and five vegetables daily. Not eating breakfast was associated with lower calcium intake in men and women, and lower iron and fibre in take in women. Achieving behavioural changes in young adults must take into account differences in dietary behaviour related to gender and SES.

Objective: To evaluate the short and long term benefits of a school and home based physical activity "enrichment" program for children at higher risk of cardiovascular disease as identified by cluster analysis., Study Design: During two 10-week school terms, 800 11-year-olds took part in a randomized controlled trial with the standard physical activity and nutrition program in six schools, the standard program in a further seven schools but with the addition of physical activity enrichment for higher risk children in those schools, and no program in five control schools. Cluster analysis identifying the 29% or so highest risk children used systolic blood pressure, percent body fat, physical fitness, and blood cholesterol., Results: Fitness improved significantly in program schools, particularly with enrichment in higher risk boys. Substantial improvements persisted 6 months later in girls from program schools. At "Enrichment" schools, cholesterol showed significant benefits in higher risk girls and, 6 months later, in both boys and higher risk girls. Sodium intake and, in girls, subscapular skinfolds were lower in "Enrichment" schools when the program ended, but not 6 months later., Conclusion: Two-semester health programs with physical activity enrichment for higher risk children can produce benefits sustained for at least 6 months. Improvements extend to lower risk children exposed indirectly to the enrichment. Attenuation of effects on diet and body composition in the longer-term suggest the need for on-going programs.

High fat diets increase body fat stores. The following experiment was undertaken to determine whether the type of dietary fat could influence fat storage and whether voluntary exercise could prevent diet-induced obesity in mice fed high fat diets. Sixty-nine 6-wk-old female mice were fed one of three diets: low fat (11.5% of energy from fat), beef fat (40.8% of energy from fat) or canola oil (40.8% of energy from fat). In each diet group, 13 mice had free access to activity wheels in their cages (exercising), and the remaining 10 mice were housed in standard mouse cages (nonexercising). Body weight and body composition were measured before and after 8 wk of treatment. The nonexercising mice fed beef fat weighed more and had significantly more body fat (23.2 +/- 2.5 g/100 g body wt) than mice fed the low fat or canola oil diet (13.9 +/- 1.7 and 16.8 +/- 1.9 g/100 g body wt, respectively). Voluntary exercise did not affect lean body mass but did result in significantly lower body fat in all diet groups (beef, 12.6 +/- 0.9; low fat, 7.4 +/- 0.6; canola oil, 9.6 +/- 1.4 g/100 g body wt). The amount of body fat of mice fed the monounsaturated canola oil was significantly less than that of mice fed the beef fat diet, suggesting that the type of fat as well as the amount of fat influences body fat stores. Furthermore, voluntary exercise decreased body fat in all mice and prevented diet-induced obesity in mice fed diets high in fat.

Myonuclear apoptosis is an early event in the pathology of dystrophin-deficient muscular dystrophy in the mdx mouse. However, events that initiate apoptosis in muscular dystrophy are unknown, and whether elimination of apoptosis can ameliorate subsequent muscle wasting remains a major question. We have tested the hypothesis that cytotoxic T-lymphocytes initiate myonuclear apoptosis in dystrophic muscle, and examined whether perforin-mediated cytotoxicity plays a role in the pathophysiology of muscular dystrophy. Mdx mice showed muscle invasion by cytotoxic T cells and helper T cells at the onset of histologically detectable muscle fiber pathology. At this time, perforin-expressing cells were also present at elevated concentration. Mdx mice depleted of CD8(+) cells showed a significant reduction of apoptotic myonuclei concentration and a reduction in necrosis, judged by macrophage invasion of muscle fibers. Double-mutant mice, deficient in dystrophin and perforin, showed nearly complete absence of myonuclear apoptosis, and a significant reduction in the concentration of macrophages in the connective tissue surrounding muscle fibers. However, muscle fiber invasion by macrophages was not reduced significantly in double mutant mice. Thus, cytotoxic T-lymphocytes contribute significantly to apoptosis and necrosis in mdx dystrophy, and perforin-mediated killing is primarily responsible for myonuclear apoptosis.

Antibodies that recognise the muscle-specific calpain 3 (CANP3) were used in a 'blind' study to label blots of skeletal muscle from 12 control subjects and from 12 patients with various muscle diseases. Calpain 3 was clearly detected in all control muscle samples analysed, even though some of the muscle had been at room temperature for over an hour before being dissected and snap-frozen. Calpain 3 was also detected in the muscle biopsies from non-LGMD2A patients, but was absent in samples from 3 patients with LGMD2A. These results show that (i) calpain 3 protein can be detected in whole extracts of human muscle, and (ii) that antibodies can be used to differentiate patients with LGMD2A from those with other muscle diseases. This represents an invaluable diagnostic aid since the limb-girdle dystrophies are very difficult to separate on clinical grounds alone. One possible function that was considered for calpain 3 was the post-translational cleavage of the 97 kDa dystroglycan precursor polypeptide into the mature alpha- and beta-dystroglycan proteins. The beta-dystroglycan band was the correct size on blots of LGMD2A muscle, indicating that calpain 3 is probably not involved in the post-translational processing of dystroglycan.

In the present investigation, we have tested the hypothesis that calpain expression or activity in skeletal muscle is influenced by changes in mechanical loading in vivo. Muscle unloading for 10 days produced no change in the concentrations of calpain I, or II, and no change in calpain activation, as assessed by measurements of the proportion of calpain I or II isoforms that exhibited autoproteolytic modifications. However, muscle reloading for 2 days produced a 90% increase in calpain II concentration per unit wet weight of muscle relative to ambulatory controls. Although no change in the activation index for calpain I or II was identified for reloaded muscle, this index is an expression of the proportion of the total mass of each calpain isoform that is autoproteolyzed. Thus, there is also approximately a 90% increase in autolyzed calpain II in muscle experiencing increased loading than in controls. Northern analysis shows that the concentration of mRNA for calpain II is increased in reloaded muscle, but no change in calpain II mRNA concentration in unloaded muscle. In situ reverse transcription polymerase chain reaction was used to confirm that nearly all calpain II mRNA in reloaded muscle is located in muscle fibers, with very little detectable calpain II mRNA in non-muscle cells present in the tissue. Together, these findings show that increased muscle loading causes a selective increase in the expression of calpain II isoform, thereby indicating that its regulation is independent from other calpain isoforms.

Neuronal nitric oxide synthase (nNOS) in fast-twitch skeletal muscle fibers is primarily particulate in contrast to its greater solubility in brain. Immunohistochemistry shows nNOS localized to the sarcolemma, with enrichment at force transmitting sites, the myotendinous junctions, and costameres. Because this distribution is similar to dystrophin, we determined if nNOS expression was affected by the loss of dystrophin. Significant nNOS immunoreactivity and enzyme activity was absent in skeletal muscle tissues from patients with Duchenne muscular dystrophy. Similarly, in dystrophin-deficient skeletal muscles from mdx mice both soluble and particulate nNOS was greatly reduced compared with C57 control mice. nNOS mRNA was also reduced in mdx muscle in contrast to mRNA levels for a dystrophin binding protein, alpha 1-syntrophin. nNOS levels increased dramatically from 2 to 52 weeks of age in C57 skeletal muscle, which may indicate a physiological role for NO in aging-related processes. Biochemical purification readily dissociates nNOS from the dystrophin-glycoprotein complex. Thus, nNOS is not an integral component of the dystrophin-glycoprotein complex and is not simply another dystrophin-associated protein since the expression of both nNOS mRNA and protein is affected by dystrophin expression.

Previous studies have shown that calpains are autolytically cleaved during the disease process of mdx dystrophy, a mouse model for Duchenne muscular dystrophy, indicating that calpains may be activated and play a role in proteolysis that occurs in muscular dystrophy (J. Biol. Chem. 270(18), 10909-10914, 1995). In the present study, we investigated the location of calpain in dystrophic muscle fibers over the course of mdx dystrophy, to relate the protease distribution to its state of activation, and to determine whether calpain translocation was an early event in mdx dystrophy. Immunolabeling of health, fully differentiated muscle fibers showed calpain present throughout the cytosol, but more concentrated near the plasma membrane. However, degenerating mdx fibers did not contain higher concentrations of calpain at the plasma membrane and showed only a homogeneous, cytosolic distribution. Calpain distribution was similarly diffuse in young myotubes and regenerating fibers with increased cytosolic concentration in early myotubes. Calpain distribution in adult mdx tissue was similar to that occurring in healthy, fully differentiated fibers, although adult mdx fibers displayed higher concentrations of membrane-associated calpain than those observed in C57 controls. The association of calpain with the plasma membrane was verified by immunoblots of isolated sarcolemmal membrane from adult mdx and control muscle which showed calpain present predominantly in the cytosol along with some membrane association. Thus, changes in calpain distribution coincide with changes in enzymatic cleavage over the course of mdx dystrophy shown previously. Furthermore, the stages of pathology at which calpain cleavage is least coincides with those stages when calpain is most concentrated at the cell membrane, suggesting that calpain is retained in an inactive form at the plasma membrane.

During cutaneous wound repair, keratinocytes move laterally across the wound surface. For this lateral movement epidermal cells must disassemble their tenacious connections to the basement membrane and their neighbouring cells, and express surface receptors that permit translocation over the wound surface extracellular matrix. If the basement membrane is disrupted, the epidermis migrates over a provisional matrix that contains fibrinogen, fibronectin, vitronectin and tenascin. Although alpha 5 beta 1 integrin, a fibronectin receptor, is expressed by human epidermis during re-epithelialization of excisional and incisional wounds, the spatial and temporal patterns of vitronectin, tenascin, and other fibronectin receptors are less clear. Other potential receptors include alpha v beta 5 for vitronectin and alpha v beta 6 for fibronectin and tenascin. To study provisional matrix integrin expression during human wound healing, full-thickness 4-mm punch biopsies were performed on the inner surface of the upper arm in adult volunteers. At 3, 7 and 14 days after injury wound sites were excised, bisected, quick frozen in liquid nitrogen, and examined for the expression of alpha 5, beta 1, alpha v, beta 5 and beta 6. At 3 days, alpha 5 beta 1 and alpha v beta 5, but not alpha v beta 6, appeared around the basal and suprabasalar cells of the migrating epidermis. At 7 days, alpha 5 beta 1, alpha v beta 5, and alpha v beta 6 appeared around the perimeter of the basal cells of the migrating epidermis. By 14 days, when re-epithelialization was complete, all basal and suprabasalar cells overlying the wound expressed alpha 5 beta 1 and alpha v beta 6, but not alpha v beta 5. Thus, alpha v appeared to switch its heterodimeric association from beta 5 to beta 6 subunit during re-epithelialization.

Calcium influx into SH-SY5Y human neuroblastoma cells after ionophore treatment or transient permeabilization in calcium-containing medium increased ALZ-50 immunoreactivity markedly. This increase was prevented by inhibitors active against calpain or against protein kinase C (PKC), suggesting that both of these enzymes were required to mediate the effect of calcium influx on ALZ-50 immunoreactivity. Treatment with PKC activator TPA increased ALZ-50 immunoreactivity in the absence of calcium influx or after intracellular delivery of the specific calpain inhibitor calpastatin, indicating that the influence of PKC was downstream from that of calpain. Calcium influx also resulted in mu-calpain autolysis (one index of calpain activation) and the transient appearance of PKM (i.e., free PKC catalytic subunits, generated by calpain-mediated cleavage of the regulatory and catalytic PKC domains). Inhibition of calpain within intact cells resulted in a dramatic increase in steady-state levels of total tau (migrating at 46-52 kDa) but resulted in a relatively minor increase in 68-kDa ALZ-50-immunoreactive tau isoforms. Although calcium influx into intact cells resulted in accumulation of ALZ-50 immunoreactivity, total tau levels were, by contrast, rapidly depleted. Incubation of isolated fractions with calpain in the presence of calcium indicated that ALZ-50-immunoreactive tau isoforms were more resistant to calpain-mediated proteolysis than were non-ALZ-50 reactive tau isoforms. These data therefore indicate that calpain may regulate tau levels directly via proteolysis and indirectly through PKC activation. A consequence of the latter action is altered tau phosphorylation, perhaps involving one or more kinase cascades, and the preferential accumulation of ALZ-50-immunoreactive tau isoforms due to their relative resistance to degradation. These findings provide a basis for the possibility that disregulation of calcium homeostasis may contribute to the pathological levels of conversion of tau to A68 by hyperactivation of the calpain/PKC system.

Diet-induced obesity was treated with a high carbohydrate, low fat diet and/or increased voluntary exercise in mice. All mice had free access to food and water during the two stage experiment. In Stage 1, 20 female mice were fed a high carbohydrate diet and 50 were made obese by consumption of a diet providing 40.8% of energy from fat. At the end of Stage 1, obese mice had significantly greater body fat stores (22.9 +/- 0.9 g/100 g body wt) than mice fed the high carbohydrate diet (12.9 +/- 1.2) (P < 0.001), yet there was no significant difference in lean body mass. In Stage 2, half of the mice were given activity wheels to increase their voluntary activity and half of the obese mice were switched to a high carbohydrate diet resulting in six groups with treatment designations of obese or lean; exercise or nonexercise, and carbohydrate or fat diets. Body fat was significantly reduced by consumption of the high carbohydrate diet (P < 0.005) and by exercise (P < 0.001), but neither treatment affected lean body mass. Exercising mice consumed significantly more energy than nonexercising mice, yet experienced a decrease in body fat and energy stores.(ABSTRACT TRUNCATED AT 250 WORDS)

We examined the interdependence of calpain and protein kinase C (PKC) activities on neurite outgrowth in SH-SY-5Y human neuroblastoma cells. SH-SY-5Y cells elaborated neurites when deprived of serum or after a specific thrombin inhibitor, hirudin, was added to serum-containing medium. The extent of neurite outgrowth under these conditions was enhanced by treatment of cells with the cell-permeant cysteine protease inhibitors N-acetyl-leucyl-leucyl-norleucinal ("C1") and calpeptin or by the phospholipid-mediated intracellular delivery of either a recombinant peptide corresponding to a conserved inhibitory sequence of human calpastatin or a neutralizing anti-calpain antisera. Calpain inhibition in intact cells was confirmed by immunoblot analysis showing inhibition of calpain autolysis and reduced proteolysis of the known calpain substrates fodrin and microtubule-associated protein 1. The above inhibitory peptides and antiserum did not induce neurites in medium containing serum but lacking hirudin, suggesting that increased surface protein adhesiveness is a prerequisite for enhancement of neurite outgrowth by calpain inhibition. Treatment of cells with the PKC inhibitor H7, staurosporine, or sphingosine induced neurite outgrowth independently of serum concentration. Because calpain is thought to regulate PKC activity, we examined this potential interrelationship during neurite outgrowth. Simultaneous treatment with calpain and PKC inhibitors did not produce additive or synergistic effects on neurite outgrowth. PKC activation by 2-O-tetradecanoylphorbol 13-acetate (TPA) prevented and reversed both neurite initiation by serum deprivation and its enhancement by calpain inhibitors. Treatment of cells with the calpain inhibitor C1 retarded PKC down-regulation following TPA treatment. Cell-free analyses demonstrated the relative specificity of various protease and kinase inhibitors for calpain and PKC and confirmed the ability of millimolar calcium-requiring calpain to cleave the SH-SY-5Y PKC regulatory subunit from the catalytic subunit, yielding a free catalytic subunit (protein kinase M). These findings suggest that the influence of PKC on neurite outgrowth is downstream from that of surface adhesiveness and calpain activity.

The current view that death of dystrophin-deficient muscle fibers is a necrotic process relies primarily upon the histological appearance of the tissue after the degenerative process is well advanced. Here, we tested this view by examining the possibility that apoptosis is a component of dystrophin-deficient muscle cell death. Three assays for apoptosis were employed in analyzing prenecrotic, peak necrotic and regenerated hindlimb muscle of mdx mice: (1) terminal deoxynucleotidyl transferase (TdT) mediated end-labeling of DNA in nuclei in tissue sections; (2) assays for DNA ladders; and (3) electron microscopic assays for the presence of organelles undergoing structural changes characteristic of apoptosis. At all ages sampled, mdx muscle contained apoptotic nuclei, according to TdT-mediated dUTP labeling of tissue sections. Nuclei in regenerated mdx muscle fibers did not display apoptosis. dUTP-labeled nuclei in control C57 muscles were rare or absent at all ages sampled. DNA from 4-week-old mdx mice was found to be cleaved into fragments indicative of preferential cleavage at internucleosomal sites. Electron microscopic analysis showed that organelle structural changes indicating apoptosis appear before pathological changes diagnostic of necrosis. For example, condensed mitochondria, fragmented sarcoplasmic reticulum and nuclei with chromatin condensations resembling apoptosis appear in fibers that otherwise possess normal morphology. Together, the findings show that apoptosis precedes any detectable necrotic change in mdx muscle, and that apoptotic events continue into the stage of dystrophic pathology that is currently viewed as necrosis. Thus, apoptosis characterizes the onset of pathology in dystrophin-deficient muscle which is followed secondarily by necrotic processes.

Death of dystrophin-deficient muscle purportedly results from increases in [Ca]in that cause the activation of calpains. We have tested whether calpains play a role in this process by assaying for changes in calpain concentration and activation in peak necrotic mdx mice (4 weeks of age) and in completely regenerated mdx mice (14 weeks of age). Biochemical fractionation and immunoblotting with epitope-specific antisera allowed measurement of the concentrations of m- and mu-calpains and the extent of autoproteolytic modification. Our findings show that total calpain concentration is elevated in both 4-week and 14-week mdx mice. This increase in concentration was shown to result primarily from a significant increase in m-calpain concentration at 4 weeks. Northern analysis demonstrated that neither m- nor mu-calpain mRNA concentrations differed between mdx and controls suggesting that the increased calpain concentration results from post-translational regulation. Immunoblotting with antibodies directed against amino-terminal peptides revealed an increase in autoproteolysis of mu-calpain, indicative of increased activation. The extent of autoproteolysis of mu-calpain returns to control levels during regeneration. This is not a consequence of increased calpastatin mRNA or protein. The findings reported here support a role for calpains in both the degenerative and regenerative aspects of mdx dystrophy.

Parathyroid hormone (PTH)-induced osteoblast retraction may play a pivotal role a role in bone resorption by providing osteoclasts direct access to mineralized bone surface. We have been working on the hypothesis that the calpains participate in this retractile response through a calcium-dependent process. We have first demonstrated the presence of calpain activities in MC3T3-E1 osteoblastic cells and that these activities can be stimulated by PTH. Second, we have demonstrated that the PTH-induced osteoblast retraction is dramatically attenuated by two different cysteine protease inhibitors. Finally, initial immunofluorescent cytochemical studies suggest that this PTH-induced osteoblastic retraction is mediated through a calpain-dependent, proteolytic modification of the cytoskeletal organization.

Calcium-activated neutral protease activity was detected in mouse MC3T3-E1 cell extracts. Inclusion of the cysteine protease inhibitor, E64c, reduced the activity, while pretreatment of intact cells with 10 nM parathyroid hormone for 90 minutes increased it. The presence of calpains in solubilized cells was confirmed by Western blotting using an antibody specific for the 80 K catalytic subunit. These results, combined with the observation that preincubation with a membrane-permeable cysteine protease inhibitor ablates 50% of the PTH-induced osteoblastic retraction, suggest that calpain-catalyzed hydrolysis of regulatory enzymes or structural proteins plays a role in mediating its short-term effects in bone.

The influence of ultraviolet B irradiation in therapeutic doses on MHC II-positive epidermal cell numbers and their surface MHC II antigen expression was studied quantitatively using light microscopic immunoperoxidase and immunogold electron microscopic techniques. In multiple ultrathin sections through many MHC II-positive epidermal cells from five healthy subjects, prior to ultraviolet exposure, Langerhans cells and indeterminate cells were found to express similar densities of surface MHC II antigens, which were uniformly distributed over the cell surface. The variation in surface MHC II antigen expression on 97 dendritic epidermal cells from one subject was normally distributed. Following a 6-week course of ultraviolet B irradiation, in the same doses as those normally used for the treatment of psoriasis, MHC II-positive epidermal cell numbers were significantly reduced (mean decrease to 51% of the pre-UVB sample; p < 0.001 analysis of variance), but their surface MHC class II antigen density was significantly increased (p < 0.05 analysis of variance). No MHC II-negative Langerhans cells were detected in either the pre- or post-UVB samples.

Dystrophin-deficient muscle undergoes sudden, postnatal onset of muscle necrosis that is either progressive, as in Duchenne muscular dystrophy, or successfully arrested and followed by regeneration, as in most muscles of mdx mice. The mechanisms regulating regeneration in mdx muscle are unknown, although the possibility that there is renewed expression of genes regulating embryonic muscle cell proliferation and differentiation may provide testable hypotheses. Here, we examine the possibility that necrotic and regenerating mdx muscles exhibit renewed or increased expression of PDGF-receptors. PDGF-binding to receptors on muscle has been shown previously to be associated with myogenic cell proliferation and delay of muscle differentiation. We find that PDGF-receptors are present in 4-week-old mdx mice in muscles that undergo brief, reversible necrosis (hindlimb muscles) or progressive necrosis (diaphragm), as well as in 4-week-old control mouse muscles. Immunoblots indicate that the concentrations of PDGF-receptors in 4-week-old dystrophic (necrotic) and control muscles are similar. Prenecrotic, dystrophic fibers and control fibers possess some cell surface labeling of fibers treated with anti-PDGF-receptor and viewed by indirect immunofluorescence. Necrotic fibers in dystrophic muscle show cytoplasmic labeling for PDGF-receptors and labeling of perinuclear regions at the muscle cell surface. Adult dystrophic muscle displays higher concentrations of PDGF-receptor in both regenerated muscle (hindlimb) and progressively necrotic muscle (diaphragm) than found in controls. Anti-PDGF-receptor labeling of regenerated, dystrophic muscle is observed primarily in granules surrounding central nuclei or surrounding nuclei located at the surface of regenerated fibers. No labeling of perinuclear regions of control muscle or prenecrotic fibers was observed. Myonuclei fractionated from adult mdx hindlimb muscles contained no PDGF-receptor, indicating that PDGF-receptor-positive structures are not tightly associated with nuclei or within nuclei. L6 myoblasts show PDGF-receptor distributed diffusely on the cell surface. Stimulation of L6 myoblasts with 10 ng/ml of PDGF-BB causes receptor internalization and concentration in granules at perinuclear regions. Thus, PDGF stimulation of myoblasts causes a redistribution of PDGF-receptors to resemble receptor localization observed during muscle regeneration. These findings implicate PDGF-mediated mechanisms in regeneration of dystrophic muscle.

The concentration, activity, and distribution of calcium-dependent proteases (calpains) are compared in dystrophin-deficient (mdx) and control mouse muscle. Calpains have been implicated previously as the protease responsible for the observed necrosis in dystrophin-deficient human muscle. Although these mouse and human muscular dystrophies have been attributed to similar genetic defects, the mouse dystrophy shows a brief necrotic episode while the human deficiency results in progressive, lethal muscle necrosis. Findings of the present study show that control mouse muscle contains more calcium-dependent proteolytic activity than dystrophin-deficient muscle. Paradoxically, adult, dystrophin-deficient mouse muscle contains higher concentrations of calpain than found in controls. Furthermore, indirect immunofluorescence using antisera produced against an oligopeptide found in the proteolytic domain of calpain shows that calpain distribution in dystrophin-deficient muscle is dispersed throughout the cytoplasm while immunolabeling of control muscle shows calpain concentrated at Z-discs. This redistribution is consistent with calpain activation in dystrophic muscle. These findings indicate that mdx mice possess the capability of suppressing calpain-mediated proteolysis. We speculate that this suppression may enable dystrophin-deficient mouse muscle to arrest necrosis and regenerate successfully.

In a recent epidemiologic survey conducted in Utah, 241 autistic patients (DSM-III criteria) were found. Medical records of 233 autistics were surveyed for the presence of 36 potentially pathologic prenatal, perinatal, and postnatal factors. These results were compared with those of an identical survey of 62 of their nonautistic siblings, with the results of four previously published surveys, and with normative data. No potentially pathologic factor or group of factors occurred significantly more frequently among the autistic patients. Also, previous observations of significant differences in the occurrence of certain factors in the histories single vs multiple siblings with autism were not confirmed, with the exception of increased viral-type illness during gestation in single-incidence cases. Thus, the etiology of the brain pathology that characteristically disrupts normal development and produces the syndrome of autism remains obscure. Other data from the epidemiologic survey, however, suggest that the role of genetic factors needs to be explored further.

The Laugier-Hunziker syndrome is an acquired, benign, macular hyperpigmentation of the lips and buccal mucosa. The nails are often involved with the development of melanonychia. Twenty-two previous cases have been recorded in the literature. We present details of six Caucasian patients with the Laugier-Hunziker syndrome who are the first recorded from Britain. They all had acquired, macular hyperpigmentation of the lips and buccal mucosa. In five of these patients longitudinal pigmented bands were found on the nails. None had other family members affected. Although this is the first report of British patients with this syndrome, we believe that the condition is probably more common than is generally recognized.

Papuloerythroderma is a newly described entity. The clinical features of a case and the therapeutic response to systemic corticosteroids are described, together with the immunohistochemical and ultrastructural characteristics of the associated inflammatory infiltrate.

Mitomycin C is an alkylating agent, used by intravesical instillation to treat carcinoma of the bladder. Repeated instillations can induce cystitis and an eczematous eruption affecting the palms, soles and face. If these effects are due to delayed hypersensitivity with sensitization to mitomycin C occurring in the bladder wall, it should be possible to demonstrate antigen-presenting cells in the bladder wall and positive patch tests to the drug. Using an immuno-alkaline phosphatase method we have identified CDI+ cells in bladder epithelium and submucosa and have demonstrated Birbeck granules in a few cells. In further support of our hypothesis it was also possible to demonstrate delayed type hypersensitivity in 13 out of 26 patients who had received mitomycin instillations by applying the allergen as a patch test. These results indicate that the eczematous eruption in this group of patients is most likely a hypersensitivity reaction and that it may be mediated transvesically.

Dientamoeba fragilis is a protozoan parasite of the large intestine of man. Individuals with infection may be asymptomatic or have gastrointestinal and systemic symptoms. We report a patient with symptomatic D. fragilis infection and negative extensive laboratory and radiological workup, with resolution of symptoms after diiodohydroxyquin therapy. No parasites were detected in three follow-up stool examinations. We then undertook retrospective study to define and describe further clinical symptoms in adults with this infection by analysis of data from medical records of 50 subjects with this parasite.

Dientamoeba fragilis is an intestinal protozoan parasite associated with gastrointestinal symptoms. This study was undertaken in a semicommunal group reported to have a high prevalence of this parasite. Stools were collected from 81 adult group members. Intestinal parasites were observed in stool specimens of 45 (56%) of the 81 adults; D. fragilis was found in 33 (41%) subjects. This paper describes the clinical findings and treatment of 26 adults with D. fragilis alone or with a commensal. Gastrointestinal symptoms were observed in 22 (85%) of infected subjects; abdominal pain and excessive flatus were significantly more common in this group. diiohydroxyquin 650 mg three times a day for 20 days eliminated the parasite in 10 (83%) of the 12 treated, although three subjects required a second course of therapy. Parasitic infection should be considered in patients with vague gastrointestinal symptoms, especially those living in endemic areas, in close proximity, or with a history of foreign travel.

Illness associated adenovirus infection is described in 15 immunocompromised patients. Patients were immunocompromised by severe underlying disease, immunosuppressive or corticosteroid therapy or by age (prematurity). Evidence of adenovirus infection was obtained by either viral isolation or, in two cases, characteristic adenovirus inclusion bodies at postmortem study. All clinical illness was associated with high fever (temperature greater than 39 degrees C). Eighty per cent of the patients had severe systemic complaints including malaise, lethargy, fatigue and night sweats; a similar number of gastrointestinal symptoms. Pulmonary complaints were described in 11 of 15 cases and included cough (67 per cent) and tachypnea (53 per cent). Roentgenologic evidence of pneumonia was demonstrated in 12 of 15 patients (80 per cent). Elevation of serum hepatic enzyme levels (serum glutamic pyruvic transaminase (SGPT)) occurred in eight of 11 patients (73 per cent) and was moderate to severe (serum glutamic pyruvic transaminase greater than 450 IU/liter) in five of 11 (45 per cent). Nine patients died; seven after a rapid downhill course and two after a prolonged illness. Evidence of adenovirus infection microscopically by autopsy in the lung, liver or both is demonstrated in four patients with fulminant systemic illness. Adenovirus infection should be considered in the etiology of severe overwhelming illness in the immunocompromised host.

To determine factors in the sexual abuse of boys, we reviewed medical records of 140 boys who were seen for evaluation at the Children's Hospital and Health Center in San Diego from 1979 to March 1984. Many of the boys were seen by one of the authors. Boys represented 9% of the 1,748 sexual abuse victims. The incidence in males increased from 7% in 1979 to 1980 to 11% in 1983. The boys were from 1 to 17 years of age. The majority of boys gave a clear, consistent, and credible history of sexual abuse. Eighty-five percent of the boys were abused by a relative or acquaintance. Physical evidence of sexual abuse was present in 95 (68%) of the victims. This paper emphasizes that boys, too, are at considerable risk for sexual abuse and that subtle physical findings of molestation are common when the child is examined by a trained, experienced physician.

A clinical trial was conducted with Alice/R-75 strain live attenuated intranasal influenza A/B vaccine. With double blind control 88 adult volunteers were administered 2 doses of Alice/R-75 vaccine, 93 volunteers received one dose of Alice/R-75 vaccine and one dose placebo solution and 94 subjects were administered 2 doses of placebo solution. Twenty-three other subjects received Alice strain monovalent influenza A vaccine. For comparison, data from 21 subjects who received monovalent intranasal R-75 strain influenza B in two doses is included. The vaccine was generally well tolerated. Four-fold serum hemagglutination-inhibiting (HAI) antibody titer rises to A/England/42/72 occurred in 39% of the monovalent Alice strain vaccines; in contrast 18% of those given 2 doses of bivalent Alice/R-75 vaccine and 11% of those given 1 dose of bivalent vaccine had similar four-fold HAI antibody titer rises. HAI antibody titer rises to influenza B/Hong Kong/72 occurred in 38% of R-75 strain monovalent vaccinees, 14% of Alice/R-75 2-dose vaccinees and 11% of Alice/R-75 one dose vaccinees. An epidemic of influenza at onset of the study made evaluation of the efficacy of the vaccine impossible.

Many vegetable foreign bodies can produce serious pulmonary complications because of chemical irritation to the airway. Barley grass, a type of grasshead, does not induce such a reaction because of its resistance to organic decay. Complications which may occur are illustrated by the clinical course of two patients with aspiration of this foreign body. In the first patient the grasshead entered the trachea with the flowering unit first and the stem following. In the second patient the stem entered the trachea first. Recurrent pneumonias were noted in the first patient. Despite its presence in the right stem bronchus for three years, no further episodes of pneumonia followed its removal. In the second patient the grassheads could not be removed endoscopically. They migrated into the right lower lobe producing pneumonia and ultimately resulting in a brain abscess. The difference of entry of the same foreign body into the trachea, stem first versus flowering unit first, is an essential factor in altering the clinical outcome.

An ice core in south Greenland covering the period 1869 to 1984 was analyzed for oxygen isotopes and chloride, nitrate, and sulfate concentrations. The data show that the "excess" (nonsea-salt) sulfate concentration has tripled since approximately 1900 to 1910 and the nitrate concentration has doubled since approximately 1955. The increases may be attributable to the deposition of these chemical specis from air masses carrying North American and Eurasian anthropogenic emissions.

Fifjty-two outpatients, aged from six months to 17 years, and suffering from Group A streptococcal pharyngitis, skin and soft tissue infection, urinary tract infection, or otitis media were enrolled in the study. The dosage of cefaclor was 20 to 40 mg/kg/day in three or four divided doses for ten days. In 43 children given either cefaclor suspension or capsules, plasma level estimations were performed. Cefaclor readily produces therapeutic plasma levels in children. Cefaclor appears to be a safe, well tolerated and effective antimicrobial agent in children. It has a distinct advantage over other antibiotics in children with Haemophilus influenzae otitis media and should be considered as a primary drug in treatment of otitis media.

We studied the frequency of parasitic infection and associated clinical symptoms in children who attended general pediatric and dental clinics at UCLA. Parasites were detected in stool specimens of 40 (38%) of the 1-4 children completing the study. Protozoan parasites were recovered in 39 (38%); one child had whipworm eggs. Parasites included Dientamoeba fragilis in 22 (21%) children and Giardia lamblia in 18 (17%) children; commensals were observed in 15 (14%) children. Parasitic infection was more frequent in younger children, those with a history of immigration or foreign travel and those attending a day care center. A significant proportion of children with parasites had vague gastrointestinal complaints; however, parasites had not been considered as the etiologic agent by the parent or child's physician. Anorexia, irritability and gas were frequent in children with G. lamblia; abdominal pain was more frequent in those with D fragilis. Intestinal parasitic infection should be considered in children with vague gastrointestional complaints, particularly those in endemic areas, with a history of foreign travel or immigration or attending a day care center. Siblings of infected children should have stool examination even if asymptomatic.

A survey was conducted to determine the prevalence of protozoan parasites in a large semicommunal group in Los Angeles. Protozoan parasites were observed in 151 (69%) of the 220 group members in the study. Parasites were observed in stool specimens from 105 (76%) of the 138 children and 46 (56%) of the 82 adults. Dientamoeba fragilis was observed in 115 (52%), Giardia lamblia in 50 (23%), Entamoeba histolytica in 9 (4%), and commensals in 61 (28%). Parasitic infection was infrequent in infants less than 1 year old, was demonstrated in 33 (89%) of the 2- to 4-year-olds, 69 (78%) of the 89 school age children 8-15 years of age, and in 46 (56%) of the 82 adults. G. lamblia was most prevalent in children younger than 6 years; whereas D. fragilis was common in all age groups. The fecal-oral route was the most likely means for parasite transmission. Since the group at times serves meals to the public, spread of parasites outside the community is a potential public health problems. Diagnosis of parasitic infection is dependent on optimal stool collection, proper laboratory techniques and trained personnel.

A retrospective study was conducted of 35 children in whom Dientamoeba fragilis was the only parasite found in the gastrointestinal (GI) tract. Gastrointestinal symptoms were present in 32 (91%) of these children; diarrhea was the most common finding in patients with acute symptoms, whereas abdominal pain was more common in children with chronic symptoms. Peripheral eosinophilia was present in half of the children examined and was statistically more significant in patients with D fragilis than in a control group of children admitted for elective surgery. Therapy with diiodohydroxyquin or metronidazole was effective; children's symptoms were diminished or were eliminated on follow-up evaluation after treatment. From this association between therapy and symptomatic relief, D fragilis should be considered pathogenic in those children with GI symptoms.