Alzheimer's disease therapeutic trials: EU/US Task Force report on recruitment, retention, and methodology.

While we may not be able to find a cure for Alzheimer's disease (AD) in the near future, several drugs presently in trials have shown promise as possible modifiers of disease progression. However, we may not be able to demonstrate efficacy due to issues of recruitment, retention, site-to-site variability, and other methodological issues. It is thus incumbent on the scientific community to find solutions to these problems, particularly as the field moves toward preventing illness or treating the disease in its prodromal stages, where these methodological issues will become even more critical. We need to better understand why participants agree or refuse to enter drug trials, and why both primary care physicians and Alzheimer's specialists agree or refuse to involve their patients. We also need to quantify the impact of requiring imaging studies, extensive questionnaires, cognitive testing, and lumbar punctures on recruitment and retention. With these concerns in mind, an international task force meeting of experts from academia and industry in the United States, European Union, and Japan in San Diego, California on November 2, 2011 to focus on recruitment, retention and other methodological issues related to clinical trials for AD. Based on the recommendations of this Task force meeting, this Perspectives article critically reflects on the most critical and timely methodological issues related to recruitment and retention in prevention and therapeutic trials in AD, which are paralleled by a paradigm shift in the diagnostic conceptualization of this disease, as reflected by recently new proposed diagnostic criteria involving preclinical stages of the disease.

Anabolic steroids induce injury and apoptosis of differentiated skeletal muscle.

Apoptosis is an active form of cellular death, or suicide, which plays an important physiologic role during organ development and in cellular turnover in differentiated tissues. Apoptosis has also been demonstrated to occur in several organs in response to hypoxic/ischemic, oxidative, or drug-induced injury and is thus involved in disease pathogenesis. However, it is generally assumed that apoptosis does not occur in differentiated skeletal muscle. Apoptosis has been demonstrated in differentiated myocardial muscle, neonatal skeletal muscle, and skeletal myoblasts in response to injury. We therefore studied differentiated murine C2 skeletal muscle cells that have been injured by supraphysiologic doses (>10 microM) of an anabolic steroid, stanozolol. Stanozolol-injured muscle cells exhibited pathologic features suggestive of apoptosis: cytoplasmic shrinkage and chromatin condensation. Muscle cells also showed positive in situ nick-end labeling of nuclear chromatin, indicating DNA strand breakage. Staining with the DNA-binding dye 33342 (bisbenzimide) also showed chromatin changes characteristic of apoptotic nuclei. Total protein levels measured at 4 and 24 hr post-stanozolol injury was not significantly decreased, indicating absence of cell lysis. Cellular ATP levels (nmol ATP/mg protein) of stanozolol-injured muscle cells, measured 4 and 24 hr postinjury, also did not change significantly. In contrast, necrotic muscle cells, injured by the calcium ionophore A23187 (2 microM), showed a progressive decline in total protein and ATP levels. This study supports two other histologic studies that showed evidence of apoptosis in differentiated skeletal muscle fibers. Our data further suggest that during the early stages of apoptosis, but not necrosis, cellular energy metabolism is preserved.

Cholinergic stimulation of skeletal muscle cells induces rapid immediate early gene expression: role of intracellular calcium.

Many properties of skeletal muscle are closely regulated by motor nerves. We have shown that nerve stimulation in vivo induced a rapid rise in mRNA for the immediate early gene (IEG) zif268 in stimulated muscle. However, the mechanisms leading to neural regulation of zif268 gene expression in muscle are not yet known. In this study, we used a mammalian skeletal muscle cell line (C2C12) to analyze the role of cholinergic transmission, and calcium flux, in the neural regulation of zif268. Treatment of the C2C12 muscle cells with carbachol, a cholinergic agonist, induced zif268 gene expression rapidly and transiently. This effect was blocked by alpha-bungarotoxin (alpha-BuTx), which specifically blocks nicotinic acetylcholine receptors. Treatment with ryanodine or dantrolene, which block the calcium release channel of the sarcoplasmic reticulum, inhibited the carbachol-induced zif268 response essentially completely. Calcium influx produced by A23187, a calcium ionophore, induced an increase of zif268 gene expression equivalent to the effect of carbachol stimulation. These results suggest that the effect of neural stimulation on zif268 may be attributable to cholinergic transmission, and the subsequent release of calcium from the sarcoplasmic reticulum.

Nerve stimulation and denervation induce differential patterns of immediate early gene mRNA expression in skeletal muscle.

Many properties of skeletal muscle cells are closely regulated by motor nerves. Neuromuscular synaptic transmission (including the 'activity' it triggers) mediates many of these effects, while denervation results in a different spectrum of muscle cell changes. However, little is known about the early regulatory events that occur in mature muscle cells in response to muscle activity or denervation. We have examined the effects of motor nerve stimulation and denervation on the expression of 4 immediate early genes (IEGs)--c-jun, junB, zif268, and nur77--in mature mouse gastrocnemius muscle. Electrical stimulation of the sciatic nerve in a pattern of brisk intermittent exercise induced a marked rise in zif268 and c-jun mRNA levels within 45 min, a minimal rise in junB, and no change in nur77 mRNA levels. By contrast, surgical denervation resulted in a marked increase of c-jun, a slight rise in junB, and no change in nur77 or zif268 mRNA levels. These findings show that neural stimulation and denervation lead to differential patterns of IEG expression. The selectivity of these patterns suggests that differential IEG expression may play an important role in regulating the specific phenotypic changes in skeletal muscles that result from denervation, innervation, and various patterns of stimulation.

Carotid high-low flow ratio most accurately predicts significant stenosis.

The Doppler quantitative carotid blood flow technique is a dynamic, physiologic tool that measures carotid blood flow, velocity, and vessel diameter. A high-low flow ratio that reflects variation in side-to-side carotid flow is calculated. We determined the accuracy of this method for the detection of carotid stenosis in 70 patients with cerebrovascular disease in whom cerebral angiography was the gold standard. The most sensitive measure proved to be high-low flow ratio, which was abnormal in 19 of 23 patients (82.6% sensitivity). The patients with high-grade stenosis had contralateral elevated flow compared with that of patients without high-grade stenosis and age-specific normal subjects. The quantitative carotid blood flow technique reflects compensatory alterations in flow produced by significant carotid disease, with an abnormal high-low flow ratio having the highest positive predictive value. This technique can be used independently to screen for carotid stenosis and as an adjuvant to carotid duplex scanning in the investigation of extracranial carotid disease.

Conduction block in diabetic neuropathy.

Symmetric sensorimotor polyneuropathy is a common complication of diabetes. Sensory and motor evoked amplitudes and conduction velocities are reduced. Both demyelination and axon loss have been reported in pathologic studies. Conduction block (CB), a manifestation of segmental demyelination, has not been previously studied in diabetic neuropathy. We determined the prevalence of conduction block in patients with diabetes by analyzing electrodiagnostic data from 24 diabetics. Conduction block was defined as a greater than 20% drop in peak-to-peak amplitude, and a less than 15% change in negative-peak duration between proximal and distal stimulation sites. A total of 76 nerve segments were studied. The criteria for conduction block were met in only 6 segments in 6 patients. The mean decrease in peak-to-peak amplitude between stimulation sites was 28% (range 21% to 40%). We conclude that conduction block over long nerve segments is uncommon in diabetic neuropathy, and, if present, suggests that other causes for neuropathy in diabetic patients should be sought.

Rapid rise in transcription factor mRNAs in rat brain after electroshock-induced seizures.

Recent studies have demonstrated that several transcription factor genes are rapidly activated by neuronal stimulation. For example, we have found that prolonged and repeated seizure activity produced by administration of chemical convulsants induces a rapid and transient increase in mRNA levels of four immediate early genes in rat brain. These genes, zif/268, c-fos, c-jun, and jun-B, encode sequence specific DNA binding proteins thought to act as transcription regulatory factors. To ascertain whether a brief electrically induced seizure discharge of the type utilized in clinical electroconvulsive treatment is sufficient to induce a similar genomic response, we have examined the response of these mRNAs in rat brain following single and repeated electroshock-induced seizures. After electroshock, mRNA levels of each of these genes increase within 15 min, and all except c-jun return to near baseline levels within 4 h. Although this response is most prominent in granule cell neurons of the hippocampus, increases are also apparent in neocortex and pyriform cortex. The rapid mRNA response persists in animals receiving a chronic electroshock protocol similar to that used in clinical electroconvulsive therapy. Intrahippocampal infusion of the sodium channel antagonist tetrodotoxin blocks hippocampal mRNA responses without blocking seizures, indicating a role for electrical excitation in the electroshock-induced mRNA response. By contrast, pretreatment with anticonvulsants or selective NMDA antagonists, which reduce seizure intensity and block hindlimb extension, fails to alter mRNA responses, suggesting that seizure induction, rather than spread, is linked to these mRNA responses. Because electroshock induces robust, highly reproducible mRNA responses, it may be useful to study the neuronal genomic response to stimulation.

Interdependence of Nitrogen Nutrition and Photosynthesis in Pisum sativum L: I. Effect of Combined Nitrogen on Symbiotic Nitrogen Fixation and Photosynthesis.

Photosynthesis, primary productivity, N content, and N(2) fixation were determined as a function of applied NH(4) (+) in peas (Pisum sativum L. cv. Alaska) which were inoculated or not inoculated with Rhizobium leguminosarum. Cabon dioxide exchange rate (CER) increased 10-fold, total N content 7-fold, and total dry weight 3-fold in 26-day-old uninoculated plants as applied NH(4) (+) was increased from 0 to 16 millimolar. In inoculated plants of the same age CER and dry weight were maximal at 2 millimolar NH(4) (+), and total N content increased between 0 and 2 millimolar NH(4) (+) but did not change significantly with higher NH(4) (+) applications. Per cent N content of uninoculated plants was significantly lower than that of inoculated plants except at the highest NH(4) (+) concentration (16 millimolar). Symbiotic N(2) fixation by inoculated plants was maximal in peas grown with 2 millimolar NH(4) (+); and apparent relative efficiency of N(2) fixation, calculated from C(2)H(2) reduction and H(2) evolution, was maximal in the 2 to 4 millimolar NH(4) (+) concentration range. The capacity to fix N(2) through the Rhizobium-legume symbiosis significantly enhanced the rate and efficiency of photosynthesis and plant N content when NH(4) (+) concentration in the nutrient solution was below 8 millimolar. Above 8 millimolar NH(4) (+) concentration uninoculated plants had greater CER, N content, and dry weight.

Interdependence of Nitrogen Nutrition and Photosynthesis in Pisum sativum L: II. Host Plant Response to Nitrogen Fixation by Rhizobium Strains.

Physiological responses to infection by strains of Rhizobium leguminosarum which differed in their capacity to reduce N(2) were determined in 26-day-old pea plants (Pisum sativum L. cv. Alaska) grown under uniform environmental conditions in the absence of combined N. The highest N(2) reduction rates, calculated from H(2) evolution and C(2)H(2)-dependent C(2)H(4) production measurements, were approximately 6-fold greater than the lowest. Higher N(2) fixation rates were associated with greater CO(2) exchange rates (R(2) = 0.92) and carboxylation efficiency (R(2) = 0.99). Increases in the apparent relative efficiency of N(2) fixation [1-(H(2) evolved in air/C(2)H(2) reduced)] (acteroid efficiency) were associated with increases in whole-plant N(2) fixation efficiency (N(2)/CO(2) reduction ratio) (R(2) = 0.95). Whole-plant dry weight and total N content were related by regression analysis (R(2) = 0.98); both parameters were enhanced by increased N(2) fixation in a manner analogous to previously reported increases caused by greater external applications of NH(4) (+). These data reveal that photosynthetic parameters in genetically uniform host plants grown under identical environmental conditions are affected by N(2) fixation characteristics of the rhizobial symbiont. The measured efficiencies of micro- and macrosymbiont are directly related under uniform environments.

Nitrogen fixation and delayed leaf senescence in soybeans.

Delayed leaf senescence has been found in a soybean population which maintains its chlorophyll and ribulosebisphosphate carboxylase activity in leaves and nitrogen fixation (acetylene reduction) activity in root nodules throughout seed maturation. Incorporation of delayed leaf senescence into an agronomically desirable genetic background may help to increase seed yield and symbiotic nitrogen fixation during seed development.