Mechanisms In Human Subjects Research Articles

Human liver-kidney model elucidates the mechanisms of aristolochic acid nephrotoxicity.

Environmental exposures pose a significant threat to human health. However, it is often difficult to study toxicological mechanisms in human subjects due to ethical concerns. Plant-derived aristolochic acids are among the most potent nephrotoxins and carcinogens discovered to date, yet the mechanism of bioactivation in humans remains poorly understood. Microphysiological systems (organs-on-chips) provide an approach to examining the complex, species-specific toxicological effects of pharmaceutical and environmental chemicals using human cells. We microfluidically linked a kidney-on-a-chip with a liver-on-a-chip to determine the mechanisms of bioactivation and transport of aristolochic acid I (AA-I), an established nephrotoxin and human carcinogen. We demonstrate that human hepatocyte-specific metabolism of AA-I substantially increases its cytotoxicity toward human kidney proximal tubular epithelial cells, including formation of aristolactam adducts and release of kidney injury biomarkers. Hepatic biotransformation of AA-I to a nephrotoxic metabolite involves nitroreduction, followed by sulfate conjugation. Here, we identify, in a human tissue-based system, that the sulfate conjugate of the hepatic NQO1-generated aristolactam product of AA-I (AL-I-NOSO3) is the nephrotoxic form of AA-I. This conjugate can be transported out of liver via MRP membrane transporters and then actively transported into kidney tissue via one or more organic anionic membrane transporters. This integrated microphysiological system provides an ex vivo approach for investigating organ-organ interactions, whereby the metabolism of a drug or other xenobiotic by one tissue may influence its toxicity toward another, and represents an experimental approach for studying chemical toxicity related to environmental and other toxic exposures.

Expression quantitative trait analyses to identify causal genetic variants for type 2 diabetes susceptibility.

Type 2 diabetes (T2D) is a common metabolic disorder which is caused by multiple genetic perturbations affecting different biological pathways. Identifying genetic factors modulating the susceptibility of this complex heterogeneous metabolic phenotype in different ethnic and racial groups remains challenging. Despite recent success, the functional role of the T2D susceptibility variants implicated by genome-wide association studies (GWAS) remains largely unknown. Genetic dissection of transcript abundance or expression quantitative trait (eQTL) analysis unravels the genomic architecture of regulatory variants. Availability of eQTL information from tissues relevant for glucose homeostasis in humans opens a new avenue to prioritize GWAS-implicated variants that may be involved in triggering a causal chain of events leading to T2D. In this article, we review the progress made in the field of eQTL research and knowledge gained from those studies in understanding transcription regulatory mechanisms in human subjects. We highlight several novel approaches that can integrate eQTL analysis with multiple layers of biological information to identify ethnic-specific causal variants and gene-environment interactions relevant to T2D pathogenesis. Finally, we discuss how the eQTL analysis mediated search for "missing heritability" may lead us to novel biological and molecular mechanisms involved in susceptibility to T2D.

Mitochondria and Endothelial Function

In contrast to their role in cell types with higher energy demands, mitochondria in endothelial cells primarily function in signaling cellular responses to environmental cues. This article provides an overview of key aspects of mitochondrial biology in endothelial cells, including subcellular location, biogenesis, dynamics, autophagy, reactive oxygen species production and signaling, calcium homeostasis, regulated cell death, and heme biosynthesis. In each section, we introduce key concepts and then review studies showing the importance of that mechanism to endothelial control of vasomotor tone, angiogenesis, and/or inflammatory activation. We particularly highlight the small number of clinical and translational studies that have investigated each mechanism in human subjects. Finally, we review interventions that target different aspects of mitochondrial function and their effects on endothelial function. The ultimate goal of such research is the identification of new approaches for therapy. The reviewed studies make it clear that mitochondria are important in endothelial physiology and pathophysiology. A great deal of work will be needed, however, before mitochondria-directed therapies are available for the prevention and treatment of cardiovascular disease.

Exome sequencing reveals FAM20c mutations associated with fibroblast growth factor 23–related hypophosphatemia, dental anomalies, and ectopic calcification

Fibroblast growth factor 23 (FGF23) plays a crucial role in renal phosphate regulation, exemplified by the causal role of PHEX and DMP1 mutations in X-linked hypophosphatemic rickets and autosomal recessive rickets type 1, respectively. Using whole exome sequencing we identified compound heterozygous mutations in family with sequence similarity 20, member C (FAM20C) in two siblings referred for hypophosphatemia and severe dental demineralization disease. FAM20C mutations were not found in other undiagnosed probands of a national Norwegian population of familial hypophosphatemia. Our results demonstrate that mutations in FAM20C provide a putative new mechanism in human subjects leading to dysregulated FGF23 levels, hypophosphatemia, hyperphosphaturia, dental anomalies, intracerebral calcifications and osteosclerosis of the long bones in the absence of rickets.

Use of laryngeal high-speed videoendoscopy systems to study voice production mechanisms in human subjects

Advances in laryngeal high-speed videoendoscopy (HSV) are making it possible to investigate critical relationships between vocal fold physiology and acoustic voice production in human subjects. Our group has developed HSV systems for clinical research with synchronous acquisition of acoustics, electroglottography, neck skin acceleration, and, in the most comprehensive setup, airflow and intraoral pressure. Key results will be presented from examinations of source-filter coupling and studies of the acoustic impact of vocal fold vibratory asymmetry in subjects with and without voice disorders. Findings hold potential clinical significance by revealing acoustic-HSV relationships not observable using standard stroboscopic imaging, as well as contributing to the direct evaluation and eventual improvement of voice production models. The work of T. F. Quatieri was supported by the Department of Defense under Air Force contract FA8721-05-C-0002. The work of other authors was supported by grants from the NIH National Institute on Deafness and Other Communication Disorders (T32 DC00038 and R01 DC007640) and by the Institute of Laryngology and Voice Restoration.

The Relationship between Levels of Greenery and Landscaping at Track and Field Sites, Anxiety, and Sports Performance of Collegiate Track and Field Athletes

Researchers wonder what it takes to improve athlete performance. Research has suggested that plants reduce anxiety, and reduced anxiety could, in turn, improve athletic performance. Research also shows that plants have psychological and restorative value such as improving coping mechanisms in human subjects as well as the potential to improve concentration and focus attention that could affect performance of athletes. The main objective of this research was to investigate the impact of greenery/landscaping on athletic performance and cognitive and somatic anxiety in track and field athletes. Four university track and field teams and 128 athletes participated in the study. Individual athlete performance and athletes' scores on the competitive state anxiety inventory-2 (CSAI-2) cognitive and somatic anxiety tests were collected from seven track meets that occurred during one spring competition season. Greenness/landscaping level was determined by Likert scale rating averages from professional horticulturists who individually rated each site. A regression analysis found that greenness level was a predictor (P = 0.000) of best performance by athletes. More of the athletes' best performance marks were at the track and field site that had the highest greenery rating, and many of the athletes' worst performance marks were achieved at the site that had the lowest greenery rating. Results also indicated that all athletes performed better at the more vegetated track and field site regardless of event and level of anxiety. All athletes performed similarly at each of the track and field sites regardless of ethnicity, gender, or grade classification. However, the overall average mean anxiety scores for all the athletes involved in this study were somewhat high in comparison with the instrument-normed scores for both the cognitive and somatic anxiety scales.

Reproducible Community Dynamics of the Gastrointestinal Microbiota following Antibiotic Perturbation

Shifts in microbial communities are implicated in the pathogenesis of a number of gastrointestinal diseases, but we have limited understanding of the mechanisms that lead to altered community structures. One difficulty with studying these mechanisms in human subjects is the inherent baseline variability of the microbiota in different individuals. In an effort to overcome this baseline variability, we employed a mouse model to control the host genotype, diet, and other possible influences on the microbiota. This allowed us to determine whether the indigenous microbiota in such mice had a stable baseline community structure and whether this community exhibited a consistent response following antibiotic administration. We employed a tag-sequencing strategy targeting the V6 hypervariable region of the bacterial small-subunit (16S) rRNA combined with massively parallel sequencing to determine the community structure of the gut microbiota. Inbred mice in a controlled environment harbored a reproducible baseline community that was significantly impacted by antibiotic administration. The ability of the gut microbial community to recover to baseline following the cessation of antibiotic administration differed according to the antibiotic regimen administered. Severe antibiotic pressure resulted in reproducible, long-lasting alterations in the gut microbial community, including a decrease in overall diversity. The finding of stereotypic responses of the indigenous microbiota to ecologic stress suggests that a better understanding of the factors that govern community structure could lead to strategies for the intentional manipulation of this ecosystem so as to preserve or restore a healthy microbiota.

Muscle tenderness from exercise: mechanisms?

When we carry out a bout of intense exercise we can, at times, reach the point where the exercise becomes painful. But as soon as we stop, the pain subsides. However, there is one form of exercise, eccentric exercise, where there is typically no pain immediately after the exercise, but we find ourselves stiff and sore next day. The soreness can persist for 4–5 days, depending on the severity of the exercise. The reason for the delayed soreness is that, in someone unaccustomed to eccentric exercise, it leads to localized areas of damage in muscle fibres. The present-day view is that the inflammatory response triggered by the damage leads to a sensitization of muscle nociceptors. For a review, see Proske & Morgan (2001). The soreness, referred to as delayed onset muscle soreness (DOMS), has a number of features that distinguish it from other forms of muscle pain. Incidentally, the accompanying sensation of muscle stiffness is the result of a damage-related rise in passive tension within the muscle (Whitehead et al. 2001). There is also some muscle swelling the day after the exercise. A characteristic feature of DOMS is that there is no chronic pain. So on the morning after the exercise we feel fine, until we get out of bed and take the first few tottering steps. Pain from DOMS can be evoked by muscle contraction, stretch and palpation, all mechanical stimuli that do not evoke any sensations of pain in an unexercised individual. It is for that reason it should be referred to as a tenderness, rather than a soreness. It has led to the view that DOMS is a type of hyperalgesia and is distinct from other kinds of muscle pain such as myositis, where there is typically some chronic pain associated with tonic activity in nociceptors (Berberich et al. 1988). Because of the unusual features of DOMS and because of the debilitating effects DOMS may have on the performance of competing athletes, there has been a need to find out more about the underlying neural mechanisms. Studying pain mechanisms in human subjects is fraught with difficulties, and only limited insight can ever be achieved, especially about central mechanisms. These are circumstances where animal models can play an important role. This issue of The Journal of Physiology contains the first description of an animal model suitable for the study of neural mechanisms underlying DOMS (Taguchi et al. 2005). The extensor digitorum longus (EDL) muscle of anaesthetized rats was contracted eccentrically numerous times and the animals were then allowed to recover. Interestingly, on recovery, the exercised rats showed no behavioural evidence of muscle tenderness. On day 2 after the exercise, one group of animals was subjected to periods of mechanical compression of the exercised muscle. Only in this group, not in the exercised animals that were not given compressions, was there evidence of up-regulation of c-Fos expression in the spinal dorsal horn, at the level of entry of afferents coming from EDL. The authors conclude that, given the diffuse and dull character of DOMS, it makes it likely to be a sensation mediated by C-fibre afferents. However, there is evidence that the story of DOMS is not as simple as that. It has been shown that the discomfort experienced from strong mechanical compression of unexercised human calf muscles can be reduced by superimposing 80 Hz vibration on the compression. This is probably an example of the phenomenon ‘rubbing it makes it better’. If this procedure is repeated in someone with the symptoms of DOMS, the vibration exacerbates the discomfort, rather than alleviating it (Weerakkody et al. 2003). The result raises a number of issues. Presumably as a result of the exercise, there has been a change in the central processing of the vibration-evoked afferent signals. Vibration frequencies of up to 120 Hz reduced the pain threshold (Weerakkody et al. 2003). It makes it unlikely that the afferents involved are in the C-fibre range, given the long refractory period for unmyelinated fibres. Other potential candidates for the vibration response are mechanically sensitive group III afferents (Paintal, 1960). It has also been shown that the pain threshold to mechanical pressure in someone with DOMS rises if the large muscle afferents, within the group I–II range, are blocked (Barlas et al. 2000; Weerakkody et al. 2003). It raises the possibility that large muscle afferents can contribute to DOMS and that DOMS could be seen to have features of a secondary hyperalgesia and allodynia. Direct evidence for mechanisms of this kind can now be sought in animal models of the type described by Taguchi et al. (2005).

Haloperidol Reduces Stimulant and Reinforcing Effects of Ethanol in Social Drinkers

Despite abundant preclinical support for a role of dopamine (DA) in the stimulant-like and reinforcing effects of ethanol, there have been few studies directly investigating this mechanism in human subjects. This study examined the effect of a DA antagonist, haloperidol, on the subjective stimulant-like effects of acute doses of ethanol and on ethanol reinforcement in healthy human volunteers. It was hypothesized that a low dose of the DA D2/D3 antagonist haloperidol (3 mg) would attenuate stimulant-like subjective effects of ethanol (0.75 g/kg) and reduce the number of drinks chosen during a subsequent choice phase. Seventeen healthy men and women, 21 to 35 years old, participated in four laboratory sessions conducted at 1-week intervals. During the four sessions they received, in randomized order under double-blind conditions, capsules containing haloperidol or placebo followed by three drinks containing ethanol (0.75 g/kg) or placebo, at 30-min intervals. Subjective and behavioral responses were measured before and after the beverages. After the third beverage, subjects could choose up to five additional doses of the beverage they had ingested. Haloperidol reduced the number of ethanol beverages subjects chose without altering placebo beverage choices. Haloperidol also dampened some of the subjective effects of ethanol, especially in subjects who experienced stimulation after ethanol. Haloperidol reduced stimulant-like and euphorigenic effects of ethanol in subjects who experienced stimulant effects (n = 8) but had no effect in subjects who did not experience stimulation from ethanol (n = 9). These findings suggest that DA plays a role in the stimulant-like, euphorigenic, and reinforcing qualities of ethanol in humans. However, the findings also raised new questions about the link between the subjective and reinforcing effects of ethanol.

Validity of animal models for the cholesterol-raising effects of coffee diterpenes in human subjects.

Cafestol and kahweol, coffee lipids present in unfiltered coffee brews, potently increase LDL-cholesterol concentration in human subjects. We searched for an animal species in which cafestol similarly increases LDL-cholesterol. Such an animal model could be used subsequently as a model to study the mechanism of action of cafestol and kahweol. Cafestol and kahweol increased serum lipids in African green monkeys (Cercopithecus aethiops), cebus (Cebus apella) and rhesus (Macaca mulatta) monkeys, hamsters, rats and gerbils differently from the increase in human subjects. In African green monkeys, the rise in total cholesterol was less pronounced than that in human subjects. In addition, the increase in total cholesterol was predominantly due to a rise in HDL-cholesterol rather than LDL-cholesterol. Thus, the rise in plasma lipids might illustrate the mechanism in these monkeys rather than the mechanism in human subjects. In other animal species, cafestol and kahweol did not raise cholesterol consistently. The variability in effects on serum lipids could not be explained by the mode of administration or dose of diterpenes, nor by the amount of cholesterol in the diet. In conclusion, we did not find an animal model in which cafestol and kahweol elevate plasma lipoproteins to the same extent as in human subjects. For the time being, therefore, studies on the mechanism of action should be done preferably in human subjects.

Early refractive development in humans

Over recent years much interest has been directed toward understanding the process by which refractive errors develop, how this is controlled and the effect of refractive errors on subsequent visual status. There are obvious difficulties in studying such mechanisms in human subjects and for this reason many studies have employed animal models. The present paper compiles the evidence available from human subjects, examining the significance of different aspects of refractive status, normal and abnormal patterns of development and their significance in the development of normal binocular function.

Muscarinic cholinergic influences on ACTH and β-endorphin release mechanisms in human subjects

Arecoline, a muscarinic cholinergic agonist, produced significant elevations in plasma cortisol and β-endorphin immunoreactivity in volunteer subjects.

Electrophysiologic and radiocinematographic study of bile flow mechanism in human subjects

PTMC, one of the choleretic, as is well known, shows a considerable serum cholesterollowering activity. In order to study this effect, th is paper will descr ible results in the effect of PTMC on hepat ic cholesterol biosynthesis and fecal excret ion of bile acids and sterols. Male mice weighing about 20g were injected for 12 consecutive days wi th PTMC (100mg/ kg/day). One hour p r io r to sacrifice on the 16 days, the animals were injected with 5 Vc of acetate-l-~4C of 0.12t~c of dl-mevalonic acid-2-14C per 20g of body weight. The liver were excised. Cholesterol was isolated as the digitonide and the fat ty acids extracted with petroleum ether, after alkaline digest was acidified. Radioactivity measurements were carried out in gas flow counter. Male ra ts weighing about 90g were used. The two-week exper imental period was divided into three groups: Unt rea ted control, cholesterol fed (100mg/day) and cholesterol f e d § (100mg/kg/day) t rea ted group. Feces were analyzed for bile acids and sterols by par t i t ion chromatography. PTMC resulted in an inhibit ion of cholesterol and fat ty acid synthesis from acetate-ln4C. Futher-more the incorporat ion of mevalonic acid-2-~*C into cholesterol was inhibited. PTMC reduced hepatic and serum cholesterol. The adminis t ra t ion of PTMC in cholesterol fed ra ts for 14 days is followed by an increase in the fecal excre t ion of bile acid, while there was no change in fecal excret ion of sterols. It is concluded tha t the mechanism of action of PTMC in lowering serum cholesterol is regard to an inhibi t ion on the biosynthesis of cholesterol and the increased el imination of bile acids with the feces.