Will biomarker-based diagnosis of Alzheimer’s disease maximize scientific progress? Evaluating proposed diagnostic criteria

A recently published framework for the diagnosis of Alzheimer’s disease (AD) in research studies would allow diagnosis on the sole basis of two biomarkers (β-amyloid and pathologic tau), even in people with no objective or subjective memory or cognitive changes. This revision will have substantial implications for future Alzheimer’s research, and the changes should be rigorously evaluated before widespread adoption. We propose three principles for evaluating any revision to diagnostic frameworks for AD: (1) does the revision improve the validity of the diagnosis; (2) does the revision improve the reliability or reduce the expense of the diagnosis; and (3) will the revision foster innovative and rigorous research across populations. The new diagnostic framework is unlikely to achieve any of these goals. Instead, it has the potential to handicap future researchers, and slow progress towards identifying effective strategies to prevent or treat AD.     

The combination of cardiorespiratory fitness and muscle strength,and mortality risk

Little is known about the combined associations of cardiorespiratory fitness (CRF) and hand grip strength (GS) with mortality in general adult populations. The purpose of this study was to compare the relative risk of mortality for CRF, GS, and their combination. In UK Biobank, a prospective cohort of >?0.5 million adults aged 40–69 years, CRF was measured through submaximal bike tests; GS was measured using a hand-dynamometer. This analysis is based on data from 70,913 men and women (832 all-cause, 177 cardiovascular and 503 cancer deaths over 5.7-year follow-up) who provided valid CRF and GS data, and with no history of heart attack/stroke/cancer at baseline. Compared with the lowest CRF category, the hazard ratio (HR) for all-cause mortality was 0.76 [95% confidence interval (CI) 0.64–0.89] and 0.65 (95% CI 0.55–0.78) for the middle and highest CRF categories, respectively, after adjustment for confounders and GS. The highest GS category had an HR of 0.79 (95% CI 0.66–0.95) for all-cause mortality compared with the lowest, after adjustment for confounders and CRF. Similar results were found for cardiovascular and cancer mortality. The HRs for the combination of highest CRF and GS were 0.53 (95% CI 0.39–0.72) for all-cause mortality and 0.31 (95% CI 0.14–0.67) for cardiovascular mortality, compared with the reference category of lowest CRF and GS: no significant association for cancer mortality (HR 0.70; 95% CI 0.48–1.02). CRF and GS are both independent predictors of mortality. Improving both CRF and muscle strength, as opposed to either of the two alone, may be the most effective behavioral strategy to reduce all-cause and cardiovascular mortality risk.     

Individually-matched etiologic studies: classical estimators made new again

With greater access to regression-based methods for confounder control, the etiologic study with individual matching, analyzed by classical (calculator) methods, lost favor in recent decades. This design was costly, and the data sometimes mis-analyzed. Now, with Big Data, individual matching becomes an economical option. To many, however, conditional logistic regression, commonly used to estimate the incidence density ratio parameter, is somewhat of a black box whose output is not easily checked. An epidemiologist-statistician pair recently proposed a new estimator that is easily applied to data from individually-matched series with a 2:1 ratio (and no other confounding variables) using just a hand calculator or spreadsheet. Surprisingly—or possibly not—they overlooked classical estimators developed in earlier decades. This prompts me to re-introduce some of these, to highlight their considerable flexibility and ease of use, and to update them. Nowadays, for any matching ratio (M:1), the Maximum Likelihood result can be easily computed from data gathered under the matched design in two different ways, each using just the summary data. One is via any binomial regression program that allows offsets, applied to just M ‘rows’ of data. The other is by hand! The aim of this note is not to save on computation; instead, it is to make connections between classical and regression-based methods, to promote terminology that reflects the concepts and structure of the etiologic study, and to focus attention on what parameter is being estimated.     

In vitro functional analysis of human cytochrome P450 2A13 genetic variants: P450 2A13*2, *3, *4, and *10

Humans possess three cytochrome P450 enzymes in the 2A subfamily (2A6, 2A7, and 2A13). P450 2A13 is mainly expressed in the human trachea and lung, whereas P450 2A6 is found in human liver. The P450 2A13 enzyme may be considered as the primary enzyme responsible for metabolic activation of many tobacco-specific carcinogens. Genetic variations significantly influence the toxicological consequences attributed to tobacco smoking. The aim of this study was to examine the in vitro functional activities of five P450 2A13 genetic variations (R257C, 133_134insT, R101Q, I331T, and R257C/I331T) in P450 2A13*2, *3, *4, and *10 alleles. Mutant clones were constructed and their recombinant enzymes were expressed in Escherichia coli. P450 2A13 mutants containing R257C, 133_134insT, I331T, and R257C/I331T displayed P450 holoenzyme spectra. The R101Q mutant was not apparently expressed. P450 2A13 enzymes displayed the typical type I binding spectra to coumarin and the calculated binding affinities of R257C, R257C/I331T, and 133_134insT mutants were decreased approximately three- to sevenfold. In catalytic analyses of purified mutant enzymes for coumarin and nicotine, the R257C and I331T mutants exhibited lower k_cat values with catalytic efficiencies reduced up to approximately 20%. The double mutation of R257C/I331T induced increased K_m values and diminished k_cat values that resulted in >50% decrease in catalytic efficiencies. For 133_134insT mutant, catalytic activities were not markedly saturated but the measured rates at the highest concentrations were significantly lower than those of the wild-type or other mutant enzymes. Functional analysis of these variations in P450 2A13 allelic variants may help to understand the consequences of P450 2A13 polymorphism in bioactivation of many tobacco-derived carcinogens.     

Evaluation of 4β-Hydroxycholesterol and 25-Hydroxycholesterol as Endogenous Biomarkers of CYP3A4: Study with CYP3A-Humanized Mice

Cytochrome P450 3A (CYP3A) enzymes metabolize approximately half of all drugs on the market. Since the endogenous compounds 4β-hydroxycholesterol (4β-HC) and 25-hydroxycholesterol (25-HC) are generated from cholesterol via CYP3A enzymes, we examined whether the plasma levels of 4β-HC and 25-HC reflect hepatic CYP3A4 activity by using a CYP3A-humanized mouse model, in which the function of endogenous Cyp3a was genetically replaced by human CYP3A. CYP3A-humanized mice have great advantages for evaluation of the relationship between hepatic CYP3A protein levels and plasma and hepatic levels of 4β-HC and 25-HC. Levels of CYP3A4 protein in the liver microsomes of CYP3A-humanized mice were increased by treatment with pregnenolone-16α-carbonitrile, a CYP3A inducer. Hepatic and plasma levels of 4β-HC and 25-HC normalized by cholesterol were significantly correlated with hepatic CYP3A4 protein levels. In addition, in vitro studies using human liver microsomes showed that the formation of 4β-HC was strongly inhibited by a CYP3A inhibitor, while the inhibitory effect of the CYP3A inhibition on the formation of 25-HC was weak. These results suggested that CYP3A mainly contributed to the formation of 4β-HC in human liver microsomes, whereas other factors may be involved in the formation of 25-HC. In conclusion, the in vivo studies using CYP3A-humanized mice suggest that plasma 4β-HC and 25-HC levels reflect hepatic CYP3A4 activity. Furthermore, taking the results of in vitro studies using human liver microsomes into consideration, 4β-HC is a more reliable biomarker of hepatic CYP3A activity.     

Detection of Memory B Activity Against a Therapeutic Protein in Treatment-Naïve Subjects

Bridging immunoassays commonly used to detect and characterize immunogenicity during biologic development do not provide direct information on the presence or development of a memory anti-drug antibody (ADA) response. In this study, a B cell ELISPOT assay method was used to evaluate pre-existing ADA for anti-TNFR1 domain antibody, GSK1995057, an experimental biologic in treatment naive subjects. This assay utilized a 7-day activation of PBMCs by a combination of GSK1995057 (antigen) and polyclonal stimulator followed by GSK1995057-specific ELISPOT for the enumeration of memory B cells that have differentiated into antibody secreting cells (ASC) in vitro. We demonstrated that GSK1995057-specific ASC were detectable in treatment-naïve subjects with pre-existing ADA; the frequency of drug-specific ASC was low and ranged from 1 to 10 spot forming units (SFU) per million cells. Interestingly, the frequency of drug-specific ASC correlated with the ADA level measured using an in vitro ADA assay. We further confirmed that the ASC originated from CD27⁺ memory B cells, not from CD27^?-naïve B cells. Our data demonstrated the utility of the B cell ELISPOT method in therapeutic protein immunogenicity evaluation, providing a novel way to confirm and characterize the cell population producing pre-existing ADA. This novel application of a B cell ELISPOT assay informs and characterizes immune memory activity regarding incidence and magnitude associated with a pre-existing ADA response.     

<Emphasis Type="Italic">In Vitro</Emphasis>-<Emphasis Type="Italic">In Vivo</Emphasis> Dose Response of Ursolic Acid,Sulforaphane, PEITC,and Curcumin in Cancer Prevention

According to the National Center of Health Statistics, cancer was the culprit of nearly 600,000 deaths in 2016 in the USA. It is by far one of the most heterogeneous diseases to treat. Treatment for metastasized cancers remains a challenge despite modern diagnostics and treatment regimens. For this reason, alternative approaches are needed. Chemoprevention using dietary phytochemicals such as triterpenoids, isothiocyanates, and curcumin in the prevention of initiation and/or progression of cancer poses a promising alternative strategy. However, significant challenges exist in the extrapolation of in vitro cell culture data to in vivo efficacy in animal models and to humans. In this review, the dose at which these phytochemicals elicit a response in vitro and in vivo of a multitude of cellular signaling pathways will be reviewed highlighting Nrf2-mediated antioxidative stress, anti-inflammation, epigenetics, cytoprotection, differentiation, and growth inhibition. The in vitro-in vivo dose response of phytochemicals can vary due, in part, to the cell line/animal model used, the assay system of the biomarker used for the readout, chemical structure of the functional analog of the phytochemical, and the source of compounds used for the treatment study. While the dose response varies across different experimental designs, the chemopreventive efficacy appears to remain and demonstrate the therapeutic potential of triterpenoids, isothiocyanates, and curcumin in cancer prevention and in health in general.