New strategy for the identification of squamous carcinoma antigens that induce therapeutic immune responses in tumor-bearing mice.

This study describes a new strategy for the identification of squamous carcinoma antigens tumor-associated antigens (TAA). The antigens were discovered by comparing microarrays of squamous carcinoma vaccines highly enriched for immunotherapeutic cells with non-enriched vaccines. The vaccines were prepared by transferring sheared genomic DNA fragments (25 kb) from KLN205 cells, a squamous carcinoma cell line (DBA/2 mouse origin (H-2(d)) into LM fibroblasts (C3H/He origin, H-2(k)). The transferred tumor DNA segments integrate spontaneously into the genome of the recipient cells, replicate as the cells divide and are expressed. As only a small proportion of the transfected cell population was expected to have incorporated DNA segments that included genes specifying TAA (the vast majority specify normal cellular constituents), a novel strategy was employed to enrich the vaccine for TAA-positive cells. Microarrays were used to compare genes expressed by enriched and non-enriched vaccines. Seventy-five genes were overexpressed in cells from the enriched vaccine. One, the gene for Cytochrome P450 (family 2, subfamily e, polypeptide 1) (Cyp2e1), was overexpressed in the enriched but not the non-enriched vaccine. A vaccine for squamous carcinoma was prepared by transfer of a 357 bp fragment of the gene for Cyp2e1 into the fibroblast cell line. Robust immunity, sufficient to result in indefinite survival, was induced in tumor-bearing mice immunized with cells transfected with this gene fragment.

Neurons of the human frontal cortex display apolipoprotein E immunoreactivity: implications for Alzheimer's disease.

Apolipoprotein E (apoE) is a plasma protein that regulates lipid transport and cholesterol homeostasis. In humans, apoE occurs as 3 major isoforms (apoE2, E3, and E4). Genetic evidence demonstrates an overrepresentation of the apoE epsilon 4 allele in Alzheimer's disease (AD). While apoE immunoreactivity (IR) is associated with the amyloid plaques and neurofibrillary tangles of AD, few studies have characterized the localization of apoE in normal human brains. We examined the distribution of apoE in the cerebral cortex of normal aged individuals and compared the results to clinically diagnosed and pathologically confirmed AD cases. In addition, we characterized the apoE IR in brains from high plaque non-demented (HPND) cases. We observed consistent and widespread apoE staining in cortical neurons from normal and HPND individuals. This finding was confirmed by double immunostaining which colocalized apoE with microtubule-associated protein-2, as well as low density lipoprotein receptor-related protein, an apoE receptor found on neurons. In contrast, AD brains displayed apoE IR in plaques and neurofibrillary tangles with little neuronal staining. These data clearly establish the presence of apoE in normal neurons, supporting an intracellular role for apoE. Moreover, the results suggest that this function of apoE is disrupted in AD, where apoE staining of neurons was drastically reduced.

Glucocorticoid-induced muscle atrophy prevention by exercise in fast-twitch fibers.

Exercise has been shown to be effective in preventing glucocorticoid-induced atrophy in muscles containing high proportions of type II or fast-twitch fibers. This investigation was undertaken to further evaluate this response in type IIa and IIb fibers, determined by histochemical staining for myofibrillar adenosinetriphosphatase with alkaline and acid preincubation. Steroid [cortisol acetate (CA), 100 mg/kg body wt] and exercise (running 90 min/day, 29 m/min) treatments were initiated simultaneously for 11 consecutive days in female rats. Fiber distribution and area measurements were performed in a deep and superficial region of plantaris muscle. The exercise regimen spared approximately 40% of the CA-induced plantaris muscle atrophy. In the deep region, the fiber population, which contained approximately 13% type I (slow-twitch), 24% type IIa, and 63% IIb fibers, was not affected by either treatment. In the superficial section, which consisted solely of type II fibers, the proportion of type IIa fibers was higher (27 vs. 9%, P less than 0.01) in the steroid- than in the vehicle-treated groups. Within each region, type IIa fibers were less susceptible to atrophy than type IIb fibers, and within each fiber type, the deep region had less atrophy than the superficial region. Type I fibers were unchanged by steroid treatment. For type IIa fibers, exercise prevented 100% of the atrophy in the deep region and 50% in the superficial region. For type IIb fibers, the activity spared 67 and 40% of the atrophy in these same regions, respectively. These results show that glucocorticoids are capable of changing the myosin phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of glucocorticoids and endurance training on cytochrome oxidase expression in skeletal muscle.

This investigation was undertaken to evaluate whether the mitochondrial disfunction associated with glucocorticoid treatment is expressed at the level of cytochrome-c oxidase (COX) and whether endurance training attenuates this response. Adult female rats were administered cortisol acetate (100 mg/kg body wt) or an equal volume of the vehicle solution for 11 days. Endurance training was performed by treadmill running up to 28 m/min (with intervals at 50 m/min for 2 min every 15 min), for 90 min/day, 6 days/wk, for 8-10 wk. During hormone treatments, the training animals ran every day. Exercise prevented 43-55% of the hormone-induced atrophy in various fast-twitch muscles or muscle groups. Cortisol acetate treatment produced no significant effects on COX enzyme activities or subunit mRNA content in deep red or superficial white quadriceps or mixed plantaris muscles. The levels of COX were increased as a result of training by 70-110% in plantaris and red quadriceps muscles, but no changes were seen in white quadriceps muscles. Both nuclear-encoded (COX IV) and mitochondrial-encoded (COX III) mRNAs were increased approximately twofold by the exercise program in these same muscles. These data indicate that the impaired mitochondrial functioning associated with glucocorticoids is not observed at the COX step of electron transport. The prolonged endurance-training regimen appears to induce relatively parallel increases in COX enzyme activity and mRNA expression with coordinate changes in nuclear and mitochondrial mRNAs.

Successive time courses of strength development and steroid hormone responses to heavy-resistance training.

Subjects (5 males, 5 females) performed heavy-resistance exercise at a constant stimulus 3 days/wk for 8 wk. Work rates were then increased to higher but constant levels for an additional 8 wk. Half times of the first training period were 14 and 10 days for the bench press and parallel squat, respectively. The second time course resulted in only one-third of the overall magnitude of strength increases for both exercises as the first, and the kinetics were slower in the parallel squat but similar in the bench press (half time 13 days). Skeletal muscle fiber area was significantly increased (19%) in fast-twitch fibers by the end of the second training period. Postexercise elevations in serum cortisol and prolactin were seen only in the male subjects. Because the males trained at considerably higher work loads than the females, these results imply that absolute amounts of resistance may be an essential requirement for inducing certain hormonal responses. Serum cortisol levels in males after exercise were blunted by the 5th wk of the first time course. For serum testosterone, neither resting nor higher post-exercise concentrations were different at any point of the training periods for either sex. We conclude that the strength time course results are not inconsistent with current thought on factors contributing to strength accumulation. The absence of a specific serum androgenic response with strength development may be related to the fact that the constant exercise stimulus is not conducive to establishing clear-cut hormone-strength relationships.

Glucocorticoid-receptor activation in hypertrophied skeletal muscle.

This investigation was undertaken 1) to determine whether the increased glucocorticoid-receptor binding activities, observed in hypertrophied plantaris muscles, are associated with a reduced ability to undergo receptor activation and 2) to examine whether glucocorticoid-receptor complexes in hypertrophied muscles undergo a shift in the relative distribution of the two thermally activated receptor forms (termed binder II and corticosteroid binder IB) to a distribution that is found in slow-twitch or heart muscle types. Plantaris muscles of female adrenalectomized rats, enlarged by surgical removal of synergists, were 60% heavier and had higher glucocorticoid cytosol binding (125 +/- 14 vs. 79 +/- 8 fmol/mg protein) than these muscles of controls. Activation, which was quantitated by the ability of the steroid-receptor complex to bind to DNA, was similar in overloaded and control muscles (57 +/- 2 vs. 62 +/- 4%). Diethylaminoethyl-cellulose chromatography of activated receptors showed approximately 16% of the radioactivity appearing as binder II and 38% as binder IB in both hypertrophied and control muscles. These results show that although enlarged plantaris muscles are undergoing certain fast- to slow-twitch biochemical transformations, the activated glucocorticoid-receptor distribution does not shift to that observed in slow fibers.

Myosin heavy chain turnover and glucocorticoid deterrence by exercise in muscle.

This study was undertaken to determine whether regular endurance running, of the type known to attenuate glucocorticoid-induced muscle atrophy, produces a reversal of the glucocorticoid-mediated suppression of myosin heavy chain (MHC) synthesis. Female rats were arbitrarily assigned to one of four groups. There were two sedentary groups that received either a vehicle (1% aqueous carboxymethyl cellulose) or cortisol acetate (100 mg/kg body wt) for 11 consecutive days and two exercise (treadmill running 29 m/min, 90 min/day, for 11 consecutive days) groups that received the activity simultaneously with either vehicle or steroid treatments. Protein synthesis measurements were performed by constant infusion of [3H]leucine. Fractional synthesis rates of MHC were determined from the leucyl-tRNA precursor pool, which was similar in all groups (range 2.85 +/- 0.32 to 3.51 +/- 0.43 dpm/pmol). Exercise prevented 30% of the plantaris muscle mass loss as the result of cortisol acetate treatment. MHC synthesis rates (%/day) in plantaris muscles of sedentary animals were reduced by glucocorticoid treatment to 65% (6.2/9.5) of the vehicle-treated group. Exercise did not alter this depression of MHC synthesis. The combination of exercise and glucocorticoid treatment reduced the calculated MHC breakdown rate (%/day) to 80% (-8.0/-10.1) of the rate resulting from hormone treatment alone and 60% (-8.0/-13.3) of the rate resulting from exercise alone. These results show that endurance exercise does not reverse the glucocorticoid inhibition of MHC synthesis in muscle but may act through reducing MHC breakdown.

Glucocorticoid antagonism by exercise and androgenic-anabolic steroids.

This work evaluated the anticatabolic capacity of androgenic-anabolic steroids and exercise (contractile activity) in inhibiting skeletal muscle atrophy associated with excessive levels of circulating glucocorticoids. With androgenic-anabolic steroids, most binding studies indicate that they have very low binding specificity for the glucocorticoid receptor. Androgens may interact through their own receptor to interfere with glucocorticoid functioning at the gene level, but this remains unproven. Current literature suggests that androgens do not prevent atrophy but may retard growth suppression accompanying glucocorticoid treatment. With exercise, functional overload, resistance, and endurance types of training are capable of preventing muscle atrophy from glucocorticoids. Androgen and glucocorticoid-receptor binding and glucocorticoid-receptor activation studies have, thus far, not established that atrophy prevention is mediated through the receptor. In conclusion, the role of androgenic-anabolic steroids as glucocorticoid antagonists requires further study. Study of the effects of exercise on muscle gene expression of glucocorticoid-inducible proteins is needed to gain additional understanding of this mechanism of atrophy prevention.

Exercise inhibits glucocorticoid-induced glutamine synthetase expression in red skeletal muscles.

One purpose of this study was to determine whether the suppression of glucocorticoid-induced glutamine synthetase (GS) gene expression by exercise is localized to fiber types that are known to be primarily recruited during endurance running. A second purpose examined whether denervation, which is associated with a reduction in contractile activity, would upregulate GS expression. Exercise consisted of treadmill running at 31 m/min for 12-16 wk. Glucocorticoid treatment (100 mg/kg body wt hydrocortisone 21-acetate) was administered during the last 11 days of the exercise program. Basal GS expression was lowest (GS enzyme activity, 43 +/- 3 nmol.h-1.mg protein-1; GS mRNA, 1.0 arbitrary units) in the slow-twitch red soleus, a muscle type that is known to resist glucocorticoid-induced muscle wasting, intermediate (74 +/- 10 and 1.7 +/- 0.2) in fast-twitch red quadriceps, a muscle type susceptible to atrophy, and highest (106 +/- 16 and 5.4 +/- 1.3) in fast-twitch white quadriceps, a muscle type known to be most susceptible to atrophy. Hormone treatment increased GS enzyme activity and mRNA by two- to fourfold in all muscle types. Exercise diminished GS enzyme activity and mRNA in the fast-twitch red fibers to 35-70% of sedentary control values in both basal and glucocorticoid-stimulated muscles. The running also reduced GS enzyme activity in hormone-treated slow-twitch fibers but did not alter basal or glucocorticoid-induced GS expression in fast-twitch white fibers. These results indicate that glucocorticoids induce similar relative GS expression across all muscle types, but the low absolute levels of expression in slow-twitch muscles are not related to any atrophy.(ABSTRACT TRUNCATED AT 250 WORDS)

Exercise interrupts ongoing glucocorticoid-induced muscle atrophy and glutamine synthetase induction.

This study was undertaken to determine whether regular endurance exercise is a deterrent to a developing state of muscle atrophy from glucocorticoids and to evaluate whether the contractile activity antagonizes the hormonal actions on glutamine synthetase, alanine aminotransferase, and cytosolic aspartate aminotransferase (cAspAT). Adult female rats were administered cortisol acetate (CA, 100 mg/kg body wt) or an equal volume of the vehicle solution for up to 15 days. Exercise (treadmill running at 31 m/min, 10% grade, 90 min/day) was introduced after 4 days of CA treatment, at which time plantaris and quadriceps muscle mass had been reduced to 90% of control levels. Running for 11 consecutive days prevented 40 mg of the 90-mg loss and 227 mg of the 808-mg loss that were subsequently observed in plantaris and quadriceps muscles, respectively, in the sedentary animals. Glutamine synthetase mRNA and enzyme activity were elevated threefold by glucocorticoid treatment in the deep quadriceps (fast-twitch red) muscles after 4 days. Initiating exercise completely interfered with the further hormonal induction (to approximately 5-fold) of this enzyme and, after 11 consecutive days of the exercise regimen, glutamine synthetase mRNA and enzyme activity were 58 and 68% of values from CA-treated sedentary animals. In vehicle-treated groups, basal levels of glutamine synthetase expression were also diminished by exercise to approximately 40% of the values in sedentary controls. Hormone treatment did not alter either aminotransferase enzyme activity but reduced cAspAT mRNA in fast-twitch red muscles by 50%. Exercise abolished the glucocorticoid effect on cAspAT mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Adverse effects of anabolic steroids.

Anabolic steroids are used therapeutically for various disorders and as ergogenic aids by athletes to augment strength, muscular development, and to enhance performance. There is a wide range of concomitant temporary and permanent adverse effects with steroid administration. Several well-documented adverse actions of these hormones may develop rapidly within several weeks or less (i.e. altered reproductive function) or require up to several years of steroid intake (i.e. liver carcinoma). More recent studies indicate that glucose intolerance, insulin resistance, increased cardiovascular disease risk profiles, cerebral dangers, musculoskeletal injuries, prostate cancer, psychosis and schizophrenic episodes, among others, accompany anabolic steroid intake. There is, at present, no evidence to support the claim that athletes are less susceptible to adverse effects than those individuals receiving hormone treatment in a clinical setting. Based on the available information which has accumulated primarily from cross-sectional, short term longitudinal, and case studies, there is a need: (a) to develop a comprehensive battery of specific and sensitive markers of adverse effects, particularly those that would be able to detect the onset of adverse actions; and (b) to conduct controlled long term longitudinal studies in order to fully understand the extensiveness and mechanisms involved in the occurrence of adverse effects.

Antagonism by glucocorticoids and exercise on expression of glutamine synthetase in skeletal muscle.

Chronic glucocorticoid treatment results in skeletal muscle wasting. However, if the contractile activity of muscle is increased, this effect is abated. Because the gene encoding glutamine synthetase is known to be glucocorticoid inducible, it represents an appropriate model for testing whether glucocorticoids and endurance training can exert antagonistic effects on the expression of specific genes in muscle tissue. Our data confirm that administration of hydrocortisone 21-acetate to rats produces 2.4- and 5.9-fold increases in plantaris muscle glutamine synthetase enzyme activity and mRNA, respectively. Moreover, subjecting rats to a 12- to 16-wk exercise program diminishes the basal levels of these indices of glutamine synthetase expression to approximately 60% of the values observed in sedentary controls. Endurance training produces a similar effect on plantaris muscle glutamine synthetase expression in glucocorticoid-treated animals. These data demonstrate that the therapeutic effects of exercise in counteracting muscle atrophy are associated with attenuation of expression of a glucocorticoid-inducible gene in skeletal muscle.

Iron levels modulate alpha-secretase cleavage of amyloid precursor protein.

The amyloid precursor protein (APP) is a membrane-spanning glycoprotein that is the source of beta A4 peptides, which aggregate in Alzheimer's disease to form senile plaques. APP is cleaved within the beta A4 sequence to release a soluble N-terminal derivative (APPsol), which has a wide range of trophic and protective functions. In the current study we have examined the hypothesis that iron availability may modulate expression or processing of APP, whose mRNA contains, based on sequence homology, a putative iron response element (IRE). Radiolabeled APP and its catabolites were precipitated from lysates and conditioned medium of stably transfected HEK 293 cells using antibodies selective for C-terminal, beta A4, and N-terminal domains. The relative abundance of the different APP catabolites under different conditions of iron availability was determined by quantitative densitometry after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The data show a specific effect on the production of APPsol. Using standard conditions previously established for IRE studies, it was found that iron chelation reduces APPsol production, whereas iron level elevation augments it. No changes were observed in levels of immature and mature APP holoprotein or in the C-terminal alpha-secretase derivative C83, beta A4, and p3 peptides. The specificity for modulatory changes in APPsol suggests that iron acts at the level of alpha-secretase activity. In addition to its modulatory effects, iron at very high levels was found to inhibit maturation of APP and production of its downstream catabolites without blocking formation of immature APP. The data establish a potential physiological role for iron in controlling the processing of APP.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduction of glutamine synthetase mRNA in hypertrophied skeletal muscle.

Skeletal muscle glutamine synthetase (GS) expression is reduced by endurance exercise and is increased when normal innervation is interrupted. This investigation was undertaken to determine whether GS expression is downregulated by the increased contractile activity associated with functional overload. Plantaris muscles overloaded for 30 days by synergist ablation were 70% heavier than those in sham-operated and unoperated control muscles. GS mRNA levels from hypertrophied muscles, measured by Northern and dot-blot hybridization, were reduced to 30% of controls. Changes in total RNA concentration and the proportion of poly(A)+ RNA in the total RNA pool did not account for the decline in GS mRNA. Despite reduced levels of GS mRNA, GS enzyme activity (nmol.h-1.mg protein-1) was unchanged in the hypertrophied muscles (overload, 79 +/- 5; control, 82 +/- 4). To further examine the lack of relationship between GS mRNA and enzyme activity, the concentration of glutamine, a known posttranslational modifier of GS activity, was measured. Consistent with the observed enzyme activities, muscle glutamine was unchanged in hypertrophied muscle (overload, 6.2 +/- 0.3; control, 5.8 +/- 0.4 mumol/g tissue). These results suggest that translational or posttranslational regulation, other than through alterations in glutamine concentration. may play a role in maintaining GS enzyme levels in hypertrophied muscle. Moreover, the regulation of GS activity in muscle hypertrophy may differ from the regulation with endurance training, in which changes in enzyme activity parallel changes in mRNA.

Isoform-specific binding of apolipoprotein E to beta-amyloid.

Apolipoprotein E (apoE), particularly the e4 allele, is genetically linked to the incidence of Alzheimer's disease. ApoE is present in the extracellular senile plaques and intracellular neurofibrillary tangles associated with Alzheimer's disease. In vitro, apoE has been shown to bind beta-amyloid (A beta), an amyloidogenic proteolytic product of amyloid precursor protein. To analyze the interaction of A beta and apoE, we used Western immunoblotting of human A beta-(1-40)-peptide incubated with conditioned medium from HEK-293 cells transfected with either human apoE3 or apoE4 (products of the e3 and e4 alleles, respectively) cDNA. Nonreducing SDS-polyacrylamide gel electrophoresis revealed the presence of an approximately 45-kDa complex with both A beta and apoE immunoreactivity. The level of the apoE3.A beta complex was approximately 20-fold greater than that of the apoE4.A beta complex. This apoE isoform-specific binding pattern was maintained from pH 5.0 to 9.0, from 2 min to 24 h of peptide incubation, and at concentrations of apoE from 5 to 100 micrograms/ml and of A beta from 10 microM to 1 mM. The higher level of apoE3 binding to A beta is in contrast to previously published data using purified apoE (Strittmatter, W. J., Weisgraber, K.H., Huang, D. Y., Dong, L.-M., Salvesen, G. S., Pericak-Vance, M., Schmechel, D., Saunders, A. M., Goldgaber, D., and Roses, A.D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8098-8102). Factors responsible for the isoform-specific interactions between apoE and A beta will require further study before the apparent discrepancy between these data can be reconciled.

Purification of apolipoprotein E attenuates isoform-specific binding to beta-amyloid.

Apolipoprotein E (apoE), particularly the e4 allele, is genetically linked to the incidence of Alzheimer's disease. In vitro, apoE has been shown to bind beta-amyloid (A beta), an amyloidogenic peptide that aggregates to form the primary component of senile plaques. In previous work, we demonstrated that apoE3 from tissue culture medium binds to A beta with greater avidity than apoE4 (LaDu, M. J., Falduto, M. T., Manelli, A. M., Reardon, C. A., Getz, G. S., and Frail, D. E. (1994) J. Biol. Chem. 269, 23403-23406). This is in contrast to data using purified apoE isoforms as substrate for A beta (Strittmatter, W. J., Weisgraber, K. H., Huang, D. Y., Dong, L.-M., Salvesen, G. S., Pericak-Vance, M., Schmechel, D., Saunders, A. M., Goldgaber, D., and Roses, A. D. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8098-8102). Here we resolve this apparent discrepancy by demonstrating that the preferential binding of A beta to apoE3 is attenuated and even abolished with purification, a process that includes delipidation and denaturation. We compared the A beta binding capacity of unpurified apoE isoforms from both tissue culture medium and intact human very low density lipoproteins with that of apoE purified from these two sources. The interaction of human A beta-(1-40)-peptide and apoE was analyzed by nonreducing SDS-polyacrylamide gel electrophoresis followed by Western immunoblotting for either A beta or apoE immunoreactivity. While the level of the apoE3.A beta complex was approximately 20-fold greater compared with the apoE4.A beta complex in unpurified conditioned medium, apoE3 and apoE4 purified from this medium bound to A beta with comparable avidity. Moreover, using endogenous apoE on very low density lipoproteins from plasma of apoE3/3 and apoE4/4 homozygotes, apoE3 was again a better substrate for A beta than apoE4. However, apoE purified from these plasma lipoproteins exhibited little isoform specificity in binding to A beta. These results suggest that native preparations of apoE may be a more physiologically relevant substrate for A beta binding than purified apoE and further underscore the importance of subtle differences in apoE conformation to its biological activity.

Effect of apolipoprotein E on neurite outgrowth and beta-amyloid-induced toxicity in developing rat primary hippocampal cultures.

The correlation between the epsilon 4 allele of apolipoprotein E (apoE) and Alzheimer's disease is well established. However, the role of apoE in normal as well as pathological brain processes remains unclear. We evaluated the effect of apoE treatment on development and beta-amyloid (A beta)-induced toxicity using primary cultures of developing rat hippocampal neurons. The source of apoE was conditioned media from HEK cells stably transfected with human apoE3 or apoE4 cDNA, a preparation where apoE is lipid-associated. Morphological and biochemical changes in the cultures were assessed at 1 and 3 days following low- and high-density plating with either apoE3 or E4 with or without A beta. Both apoE isoforms were neurotrophic, as measured by increased neurite length. Aged A beta(1-42), a peptide preparation exhibiting extensive fibril and aggregate formation, is toxic to these cultures. Addition of apoE3 and E4 significantly and comparably attenuated the A beta-induced reduction in both neurite length and cell viability. The level of protection against this toxicity was proportional to the neurotrophic actions of the two apoE isoforms. Thus, apoE acts as a potent growth factor in both the absence and the presence of A beta, supporting a potentially important role for apoE in neurobiology.