'Time Saved' As a Demonstration of Clinical Meaningfulness and Illustrated Using the Donanemab TRAILBLAZER-ALZ Study Findings.

In Alzheimer's disease (AD) clinical trials, disease-modifying therapies are expected to slow the rate of disease progression. Treatment effects are evaluated using a validated clinical scale as the difference between treatment and placebo in mean change from baseline to endpoint. Understanding the clinical relevance of this metric is not necessarily intuitive. Expressing active treatment-placebo difference as a time metric (i.e., months saved with treatment) has potential to provide a metric that is more easily and consistently interpreted. Using data from the TRAILBLAZER-ALZ study, time component tests (TCTs) were employed to determine the time saved with donanemab (an amyloid lowering drug) treatment. At study endpoint (Week 76), disease progression was delayed by 5.3 months and 5.2 months as measured by the Integrated Alzheimer's Disease Rating Scale (iADRS) and the Clinical Dementia Rating Sum of Boxes (CDR-SB), respectively.

A statewide outbreak of Cryptosporidium and its association with the distribution of public swimming pools.

In order to characterize the association between county-level risk factors and the incidence of Cryptosporidium in the 2007 Iowa outbreak, we used generalized linear mixed models with the number of Cryptosporidium cases per county as the dependent variable. We employed a spatial power covariance structure, which assumed that the correlation between the numbers of cases in any two counties decreases as the distance between them increases. County population size was included in the model to adjust for population differences. Independent variables included the number of pools in specific pool categories (large, small, spa, wading, waterslide) and pool-owner classes (apartment, camp, country club or health club, hotel, municipal, school, other) as well as the proportion of residents aged <5 years. We found that increases in the number of bigger pools, pools with more heterogeneous mixing (municipal pools vs. country club or apartment pools), and pools catering to young children (wading pools) are associated with more cases at the county level.

Driving under low-contrast visibility conditions in Parkinson disease.

OBJECTIVE: To assess driving performance in Parkinson disease (PD) under low-contrast visibility conditions. METHODS: Licensed, active drivers with mild to moderate PD (n = 67, aged 66.2 +/- 9.0 years, median Hoehn-Yahr stage = 2) and controls (n = 51, aged 64.0 +/- 7.2 years) drove in a driving simulator under high- (clear sky) and low-contrast visibility (fog) conditions, leading up to an intersection where an incurring vehicle posed a crash risk in fog. RESULTS: Drivers with PD had higher SD of lateral position (SDLP) and lane violation counts (LVC) than controls during fog (p < 0.001). Transition from high- to low-contrast visibility condition increased SDLP and LVC more in PD than in controls (p < 0.01). A larger proportion of drivers with PD crashed at the intersection in fog (76.1% vs 37.3%, p < 0.0001). The time to first reaction in response to incursion was longer in drivers with PD compared with controls (median 2.5 vs 2.0 seconds, p < 0.0001). Within the PD group, the strongest predictors of poor driving outcomes under low-contrast visibility conditions were worse scores on measures of visual processing speed and attention, motion perception, contrast sensitivity, visuospatial construction, motor speed, and activities of daily living score. CONCLUSIONS: During driving simulation under low-contrast visibility conditions, drivers with Parkinson disease (PD) had poorer vehicle control and were at higher risk for crashes, which were primarily predicted by decreased visual perception and cognition; motor dysfunction also contributed. Our results suggest that drivers with PD may be at risk for unsafe driving in low-contrast visibility conditions such as during fog or twilight.

Driving with distraction in Parkinson disease.

OBJECTIVE: To assess the effects of auditory-verbal distraction on driving performance in Parkinson disease (PD). METHODS: We tested licensed, currently active drivers with mild-to-moderate PD (n = 71) and elderly controls with no neurologic disease (n = 147) on a battery of cognitive, visual, and motor tests. While they drove on a four-lane interstate freeway in an instrumented vehicle, we determined at-fault safety errors and vehicle control measures during a distracter task (Paced Auditory Serial Addition Task [PASAT]) and on an uneventful baseline segment. RESULTS: Compared with controls, drivers with PD committed more errors during both baseline and distraction, and drove slower with higher speed variability during distraction. Although the average effect of distraction on driving performance compared with baseline was not different between the groups, the drivers with PD showed a more heterogeneous response to distraction (p < 0.001): the error count increased in 28.2% of drivers with PD (vs 15.8% in controls), decreased in 16.9% (vs 3.4%), and remained stable in 54.9% (vs 80.8%). The odds of increase in safety errors due to distraction was higher in the PD group even after adjusting for baseline errors, level of engagement in PASAT, sex, and education (odds ratio [95% CI] = 2.62 [1.19 to 5.74], p = 0.016). Decreased performance on tests of cognitive flexibility, verbal memory, postural control, and increased daytime sleepiness predicted worsening of driving performance due to distraction within the PD group. CONCLUSION: The quantitative effect of an auditory-verbal distracter task on driving performance was not significantly different between Parkinson disease (PD) and control groups. However, a significantly larger subset of drivers with PD had worsening of their driving safety errors during distraction. Measures of cognition, motor function, and sleepiness predicted effects of distraction on driving performance within the PD group.

The triple threat to nascent apolipoprotein B. Evidence for multiple, distinct degradative pathways.

We previously showed that Omega-3 fatty acids reduce secretion of apolipoprotein B (apoB) from cultured hepatocytes by stimulating post-translational degradation. In this report, we now characterize this process, particularly in regard to the two known processes that degrade newly synthesized apoB, endoplasmic reticulum (ER)-associated degradation and re-uptake from the cell surface. First, we found that Omega-3-induced degradation preferentially reduces the secretion of large, assembled apoB-lipoprotein particles, and apoB polypeptide length is not a determinant. Second, based on several experimental approaches, ER-associated degradation is not involved. Third, re-uptake, the only process known to destroy fully assembled nascent lipoproteins, was clearly active in primary hepatocytes, but Omega-3-induced degradation of apoB continued even when re-uptake was blocked. Cell fractionation showed that Omega-3 fatty acids induced a striking loss of apoB100 from the Golgi, while sparing apoB100 in the ER, indicating a post-ER process. To determine the signaling involved, we used wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, which blocked most, if not all, of the Omega-3 fatty acid effect. Therefore, nascent apoB is subject to ER-associated degradation, re-uptake, and a third distinct degradative pathway that appears to target lipoproteins after considerable assembly and involves a post-ER compartment and PI3K signaling. Physiologic, pathophysiologic, and pharmacologic regulation of net apoB secretion may involve alterations in any of these three degradative steps.

Insulin-treated Zucker diabetic fatty rats retain the hypertriglyceridemia associated with obesity.

Lipoprotein and apolipoprotein changes were evaluated in 10-week-old Zucker diabetic fatty (ZDF) male rats following 12 weeks of insulin treatment, which normalized blood glucose and maintained weight gaining characteristic of nondiabetic Zucker fatty rats. Compared with untreated ZDF rats (saline-injected), insulin treatment resulted in increased very-low-density lipoprotein (VLDL; d < 1.006 g/mL) and decreased alpha lipoprotein on agarose gel electrophoresis. These findings were consistent with an observed increase in VLDL triglyceride and cholesterol, and decreased high-density lipoprotein (HDL) cholesterol with insulin treatment in isolated lipoproteins. B100 levels were unchanged by insulin treatment, but B48 levels were significantly increased in the VLDL fraction. Insulin treatment depressed apolipoprotein (apo) A-I levels in HDL, but had little effect on total apo E, apo A-IV, or apo C, although apo C was redistributed to the VLDL fraction. These results suggest that insulin treatment of ZDF rats normalizes hyperglycemia and prevents age-related changes in lipoprotein parameters associated with development of insulinopenic diabetes. Insulin therapy in ZDF rats thereby sustains the hyperlipidemic lipoprotein pattern associated with hyperinsulinemia and obesity.

Glucose-stimulated insulin secretion suppresses hepatic triglyceride-rich lipoprotein and apoB production.

The current study assessed in vivo the effect of insulin on triglyceride-rich lipoprotein (TRL) production by rat liver. Hepatic triglyceride and apolipoprotein B (apoB) production were measured in anesthetized, fasted rats injected intravenously with Triton WR-1339 (400 mg/kg). After intravascular catabolism was blocked by detergent treatment, glucose (500 mg/kg) was injected to elicit insulin secretion, and serum triglyceride and apoB accumulation were monitored over the next 3 h. In glucose-injected rats, triglyceride secretion averaged 22.5 +/- 2.1 microg.ml(-1).min(-1), which was significantly less by 30% than that observed in saline-injected rats, which averaged 32.1 +/- 1.4 microg.ml(-1).min(-1). ApoB secretion was also significantly reduced by 66% in glucose-injected rats. ApoB immunoblotting indicated that both B100 and B48 production were significantly reduced after glucose injection. Results support the conclusion that insulin acts in vivo to suppress hepatic very low density lipoprotein (VLDL) triglyceride and apoB secretion and strengthen the concept of a regulatory role for insulin in VLDL metabolism postprandially.

B48 is preferentially translated over B100 in cells with increased endogenous apo B mRNA.

We recently demonstrated that expression of BHMT in McArdle RH-7777 (McA-BHMT) cells increases apo B mRNA abundance, leading to parallel increases in apo B secretion. The ratio of unedited to edited apo B mRNA was unchanged by BHMT expression. Based on the observation that secretion of B48 is increased relative to B100 in McA-BHMT cells, current studies now include comparison of B48 and B100 synthesis and intracellular degradation. Minor differences in co- and posttranslational degradation were unable to account for relative increase in B48 secretion, and the disappearance kinetics of B48 were similar in McA-BHMT and control cells. Consistent with the increase in endogenous apo B mRNA in McA-BHMT cells, B48 synthesis is increased significantly. In contrast, synthesis of B100 was not significantly increased. We conclude that B48 is preferentially translated compared to B100 when endogenous apo B mRNA is increased.

Effect of dietary fat on the development of non-insulin dependent diabetes mellitus in obese Zucker diabetic fatty male and female rats.

The obese Zucker diabetic fatty male rat (ZDF/Gmi¿trade mark omitted¿-fa) has become a widely used animal model of NIDDM, in contrast to the obese ZDF females that rarely develop NIDDM. However, preliminary observations suggest that obese ZDF females may become diabetic on high-fat diets. Therefore, we studied the effect of dietary fat on development of NIDDM, dyslipidemia, and alterations in organ-specific serum panels in obese ZDF males and females. Results indicated different effects of dietary fat-content on development of diabetes in males and females. Males, even on low fat-content diets, developed diabetes but the process was accelerated as a function of dietary fat-content, whereas only the highest fat-content diet induced development of NIDDM in obese ZDF females. Additionally, triglyceride/apolipoprotein B ratios demonstrated gender-specific differences in the nature of circulating lipoprotein particles independent of diabetic state with values for females approximately twice those of males indicating more highly triglyceride-enriched lipoprotein particles in females. We conclude that the obese ZDF female rat has the potential to become an important animal model of NIDDM especially in women where few models currently exist.

Apolipoprotein B mRNA and lipoprotein secretion are increased in McArdle RH-7777 cells by expression of betaine-homocysteine S-methyltransferase.

The cDNA encoding rat betaine-homocysteine S-methyltransferase (BHMT) was isolated through production of monoclonal antibodies against protein fractions enriched with apolipoprotein B (apo B)-mRNA-editing complexes. BHMT mRNA was expressed predominantly in liver, and also in kidney, but not in small intestine. In stable McArdle RH-7777 (McA) cell lines expressing differing levels of BHMT, the editing efficiency of apo B mRNA was unchanged. Evaluation of apo B-mRNA expression revealed that steady-state levels were increased significantly and in parallel with BHMT protein expression. The highest levels of BHMT mRNA and BHMT enzyme activity expressed in stably transfected McA cells were comparable with those found in rat hepatocytes. In contrast to the changes in apo B-mRNA abundance, levels of other apolipoprotein-encoding mRNAs and several liver-specific and ubiquitously expressed mRNAs were unchanged by BHMT expression. In the cell line expressing the highest level of BHMT, apo B-containing lipoprotein secretion was increased, indicating utilization of increased endogenous message. Results suggest that apo B-mRNA abundance in McA cells is related to the expression of BHMT, an enzyme important in homocysteine metabolism.

Lipoprotein alterations in 10- and 20-week-old Zucker diabetic fatty rats: hyperinsulinemic versus insulinopenic hyperglycemia.

Lipoprotein and apolipoprotein parameters were studied in the male Zucker diabetic fatty (ZDF) rat at 10 and 20 weeks of age, corresponding to hyperinsulinemic and insulinopenic type 2 diabetes mellitus, respectively. At both ages, ZDF rats had elevated serum triglycerides, free fatty acids, and corticosterone, whereas 20-week ZDF rats had reduced thyroid hormones. At 10 weeks, the hyperlipidemia was confined to elevations in pre-beta triglyceride-rich (d < 1.006 g/mL) lipoproteins. By 20 weeks, all lipoprotein density fractions were increased compared with lean rats, with substantial increases in both low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol. In ZDF rats, there was a progressive increase in apolipoprotein B (apo B) from 1.9 times control at 10 weeks to three times control at 20 weeks. The increase in apo B was accompanied by a shift of apo B, particularly B100, from very-low-density lipoprotein (VLDL) into denser lipoproteins corresponding to intermediate-density lipoproteins plus LDLs (1.006 < d < 1.063 g/mL). In Zucker and 10-week ZDF rats, in the presence of hyperinsulinemia, the increase in serum apo B was predominantly apo B48 present in VLDL. By 20 weeks, when ZDF rats are insulinopenic, the mass ratio of B48:B100 shifted from 2.7 to 0.7. The shift was associated with a decrease in hepatic-edited apo B mRNA. Apo E increased in lean rats between 10 and 20 weeks of age. Although apo E also increased in ZDF rats, the increase by 20 weeks was less than that of lean rats. The molar ratio of apo E to B in VLDL was decreased in ZDF rats. In lean rats, greater than 50% of apo E was present in HDL, in contrast to ZDF rats, where less than 20% of apo E was present in HDL. VLDL apo E shifted to denser fractions by 20 weeks of age, similar to apo B. The apo C level was more than double compared with the level in lean rats and was redistributed from the HDL fraction to lipoprotein fractions containing apo B. Both apo A-I and apo A-IV levels more than doubled between 10 and 20 weeks in ZDF rats. The ZDF rat model may be useful in comparative studies of lipoproteins during diabetic progression from hyperinsulinemia to insulinopenia.

Ethanol increases apolipoprotein B mRNA editing in rat primary hepatocytes and McArdle cells.

Apolipoprotein B (apoB) mRNA editing involves a site-specific cytidine to uridine transition catalyzed by the cytidine deaminase, APOBEC-1, in the context of and regulated by a multi-protein-containing editosome. ApoB mRNA editing in vivo is subject to tissue specific, developmental and metabolic regulation. We demonstrate for the first time that the amount of edited apoB mRNA in rat primary hepatocytes is markedly increased subsequent to transient treatment with ethanol in vitro. The apparent change in editing efficiency was dose-dependent (from 0.1%-2.4% initial ethanol dose) and occurred with rapid onset. The proportion of edited apoB mRNA was also markedly enhanced in a rat hepatoma cell line, McArdle RH7777 cells and in a stable McArdle cell line over-expressing APOBEC-1 by transient treatment with 2.5 % ethanol. In contrast, the apoB mRNA editing in a human hepatoma cell line, HepG2 cells and a stable HepG2 cell line over-expressing APOBEC-1 did not respond to ethanol treatment. The data support the possibility that editing activity is ethanol-responsive but suggest that this change is cell type-specific.

Suppression of the protein tyrosine phosphatase LAR reduces apolipoprotein B secretion by McA-RH7777 rat hepatoma cells.

Apolipoprotein B (apo B) secretion is reduced by insulin in rat hepatocytes. To evaluate possible mechanisms by which insulin action leads to inhibition of apo B secretion, we evaluated the effect of suppression of the protein-tyrosine phosphatase LAR on apo B secretion by McA-RH7777 (McA) rat hepatoma cells. A reduction in cellular LAR levels was accomplished by stable transfection of McA cells with LAR antisense cDNA. Previous studies indicate that LAR-antisense transfectants demonstrate increased insulin receptor signaling. In current studies, reduced LAR expression results in a 60% to 70% reduction in apo B secretion compared with null vector control. The reduction in apo B secretion correlated with a significant decrease in cellular apo B mRNA levels. Results suggests there is a relationship of protein tyrosine phosphorylation with regulation of apo B mRNA abundance in McA cells.

Effects of fatty acids on apolipoprotein B secretion by McArdle RH-7777 rat hepatoma cells.

The effect of oleic acid (OA), stearic acid (SA) and elaidic acid (EA) on cellular and secreted apolipoprotein (apo) B was examined in McArdle RH-7777 (McArdle) hepatoma cells and in primary rat hepatocytes. ApoB secretion by McArdle cells was significantly inhibited by 20% in 8 h incubations in medium containing EA and SA and by 50% in medium containing OA. In contrast, apo B secretion and cellular apo B of primary rat hepatocytes was relatively unaffected by incubations in medium containing fatty acids. Both B100 and B48 secretion in McArdle wild type and B48 in apo B mRNA editing enzyme catalytic polypeptide transfectants expressing B48 were inhibited to a similar extent indicating an effect of OA on both apo B species. The effect of OA occurred without changes in cellular apo B or in apo B mRNA abundance suggesting a post-transcriptional mechanism. Time course studies indicate that the suppressive effect of OA requires 4 h of incubation suggesting the depletion of a limiting factor important in apoB secretion. By increasing the proportion of palmitic acid to OA in the medium, apoB secretion by McArdle cells was progressively restored to control levels implicating an unique role for newly synthesized saturated fatty acid.

Phosphoinositide 3-kinase activity is necessary for insulin-dependent inhibition of apolipoprotein B secretion by rat hepatocytes and localizes to the endoplasmic reticulum.

Insulin inhibits apolipoprotein B (apoB) secretion by primary rat hepatocytes through activation of phosphoinositide 3-kinase (PI 3-K). Current studies demonstrate that the PI 3-K inhibitor wortmannin inhibits both basal and insulin-stimulated PI 3-K activities. Wortmannin and LY 294002, two structurally distinct PI 3-K inhibitors, prevent insulin-dependent inhibition of apoB secretion in a dose-dependent manner. To link PI 3-K activation to insulin action on apoB, we investigated whether insulin induced localization of activated PI 3-K to the endoplasmic reticulum (ER), where apoB biogenesis is initiated. Insulin action results in a significant redistribution of PI 3-K to a low density microsome (LDM) fraction containing apoB protein and apoB mRNA. Insulin stimulates a significant increase in PI 3-K activity associated with insulin receptor substrate-1 as well as an increase in insulin receptor substrate-1/PI 3-K mass in LDM. Subfractionation of LDM on sucrose density gradients shows that insulin significantly increases the amount of PI 3-K present in an ER fraction containing apoB. Insulin stimulates PI 3-K activity in smooth and rough microsomes isolated from rat hepatocytes, the latter of which contain rough ER as demonstrated by electron microscopy. Studies indicate that 1) PI 3-K activity is necessary for insulin-dependent inhibition of apoB secretion by rat hepatocytes; 2) insulin action leads to the activation and localization of PI 3-K in an ER fraction containing apoB; and 3) insulin stimulates PI 3-K activity in the rough ER.

Regulation of hepatic apolipoprotein B RNA editing in the genetically obese Zucker rat.

Hepatic apolipoprotein (apo) B RNA editing was examined in the genetically obese hyperinsulinemic and hypertriglyceridemic Zucker rat. In obese Zucker rats, apo B RNA editing was increased 42% relative to that in lean controls. Correspondingly, the proportion of serum triglyceride-rich lipoprotein containing apo B48 increased 4.7-fold in the obese Zucker rat. Quantification of hepatic total apo B mRNA showed no difference between obese Zucker and lean control rats. In contrast, the hepatic mRNA encoding APOBEC-1, the catalytic subunit of the RNA editing activity, demonstrated an increased abundance of 1.8-fold in obese Zucker rats versus lean controls.

An auxiliary factor containing a 240-kDa protein complex is involved in apolipoprotein B RNA editing.

A protein complex involved in apolipoprotein B (apoB) RNA editing, referred to as AUX240 (auxiliary factor containing p240), has been identified through the production of monoclonal antibodies against in vitro assembled 27S editosomes. The 240-kDa protein antigen of AUX240 colocalized with editosome complexes on immunoblots of native gels. Immunoadsorbed extracts were impaired in their ability to assemble editosomes beyond early intermediates and in their ability to edit apoB RNA efficiently. Supplementation of adsorbed extract with AUX240 restored both editosome assembly and editing activities. Several proteins, in addition to p240, ranging in molecular mass from 150 to 45 kDa coimmunopurify as AUX240 under stringent wash conditions. The activity of the catalytic subunit of the editosome APOBEC-1 and mooring sequence RNA binding proteins of 66 and 44 kDa could not be demonstrated in AUX240. The data suggest that p240 and associated proteins constitute an auxiliary factor required for efficient apoB RNA editing. We propose that the role of AUX240 may be regulatory and involve mediation or stabilization of interactions between APOBEC-1 subunits and editing site recognition proteins leading the assembly of the rat liver C/U editosome.

Insulin-mediated inhibition of apolipoprotein B secretion requires an intracellular trafficking event and phosphatidylinositol 3-kinase activation: studies with brefeldin A and wortmannin in primary cultures of rat hepatocytes.

Insulin inhibition of the secretion of apolipoprotein B (apo B) was studied in primary cultures of rat hepatocytes by using brefeldin A (BFA), an inhibitor of protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus, and by using the phosphatidylinositol 3-kinase (PI 3-K) inhibitor wortmannin. Incubation of hepatocytes with BFA (10 micrograms/ml) for 1 h inhibited the subsequent secretion of apo B, albumin and transferrin for up to 3 h. BFA treatment resulted in the time-dependent accumulation in cells of [14C]leucine-labelled proteins and apo B. Under conditions where insulin decreased total apo B (cell plus secreted), BFA blocked the insulin-dependent effect. These results suggest that export of apo B from the ER is a prerequisite for the observed insulin effect. Treatment of hepatocytes with wortmannin for 20 min abolished insulin inhibition of apo B secretion, suggesting that the insulin effect on the apo B pathway involves activation of PI 3-K. Enzyme inhibitor studies indicate that chymostatin and (+)-(2S,3S)-3-[(S)-methyl-1-(3-methylbutylcarbamoyl)-butylcarba moyl]-2- oxiranecarboxylate (E-64-c) partially block insulin effects on apo B compared with leupeptin, which had no discernible effect. The cell-permeable derivative of E-64-c, EST, and N-Ac-Leu-Leu-norleucinal (ALLN) were most effective in blocking insulin effects on apo B. These results suggest that insulin action on apo B in primary rat hepatocytes involves (1) vesicular movement of apo B from the ER; (2) activation of PI 3-K and (3) a cellular protease that is either a cysteine- or calcium-activated neutral protease.

Obese Zucker (fa/fa) rats are resistant to insulin's inhibitory effect on hepatic apo B secretion.

Hepatocytes derived from lean Zucker rats have reduced secretion of apo B and lowered cellular apo B in response to a physiologic range of insulin (0.1 nM-10 nM). Effects are attenuated in hepatocytes derived from Zucker obese rats and seen only at higher insulin concentrations (> 100 nM) with a significant shifting of the dose-response curve. Decreased sensitivity and responsiveness of hepatocytes derived from obese rats suggests insulin resistance and dose-response curves are consistent with coexistent binding and post-binding defects. Inability to inhibit hepatic apo B secretion in the presence of short-term high levels of insulin may have important implications to the balance of intestinal and hepatic triglyceride-rich lipoprotein secretion post-prandially.