Actual conditions for returning home after hospitalization among older patients receiving home medical care in Japan: OHCARE Study.

AIM: To examine the actual conditions of older patients receiving home medical care after hospitalization over a period of 2 years in Japan. METHODS: The study population included 102 participants, aged ≥65 years, receiving home medical care, who consented to participate in the Osaka Home Care Registry (OHCARE) study in Japan over a period of 2 years. We investigated the actual conditions for returning home after hospitalization. RESULTS: The median age of the 102 participants was 84 years, and 61 (59.8%) were women. In the group that returned home, 42 (55.3%) of the respondents desired to recuperate in a familiar place, as in advanced care planning (ACP). During the 2-year follow-up period, the group that did not return home had significantly more deaths. A multivariate analysis showed the association in the presence of ACP (odds ratio: 4.72, 95% confidence interval: 1.60-13.86) and cardiac disease (odds ratio: 0.25, 95% confidence interval: 0.08-0.76). The lack of ACP in the medical records when the patient was admitted to the hospital may have prevented the return home. CONCLUSION: In older patients who had difficulty returning home after hospitalization, the lack of ACP in home medical care may have been an influencing factor. ACP could help continue with home medical care. Geriatr Gerontol Int 2024; 24: 320-326.

Neural effects of acute stress on appetite: A magnetoencephalography study.

Stress is prevalent in modern society and can affect human health through its effects on appetite. Therefore, in the present study, we aimed to clarify the neural mechanisms by which acute stress affects appetite in healthy, non-obese males during fasting. In total, 22 volunteers participated in two experiments (stress and control conditions) on different days. The participants performed a stress-inducing speech-and-mental-arithmetic task under both conditions, and then viewed images of food, during which, their neural activity was recorded using magnetoencephalography (MEG). In the stress condition, the participants were told to perform the speech-and-mental-arithmetic task again subsequently to viewing the food images; however, another speech-and-mental-arithmetic task was not performed actually. Subjective levels of stress and appetite were then assessed using a visual analog scale. Electrocardiography was performed to assess the index of heart rate variability reflecting sympathetic nerve activity. The findings showed that subjective levels of stress and sympathetic nerve activity were increased in the MEG session in the stress condition, whereas appetite gradually increased in the MEG session only in the control condition. The decrease in alpha band power in the frontal pole caused by viewing the food images was greater in the stress condition than in the control condition. These findings suggest that acute stress can suppress the increase of appetite, and this suppression is associated with the frontal pole. The results of the present study may provide valuable clues to gain a further understanding of the neural mechanisms by which acute stress affects appetite. However, since the stress examined in the present study was related to the expectation of forthcoming stressful event, our present findings may not be generalized to the stress unrelated to the expectation of forthcoming stressful event.

Neural effects of hand-grip-activity induced fatigue sensation on appetite: a magnetoencephalography study.

It has been reported that physical activity not only increases energy expenditure, but also affects appetite. However, little remains known about the effects of physical activity-induced fatigue sensation on appetite. In the present study, classical conditioning related to fatigue sensation was used to dissociate fatigue sensation from physical activity. The participants were 20 healthy male volunteers. After overnight fasting, on day 1, the participants performed hand-grip task trials for 10 min with listening to a sound. The next day, they viewed food images with (target task) and without (control task) listening to the sound identical to that used on day 1, and their neural activity during the tasks were recorded using magnetoencephalography. The subjective levels of appetite and fatigue sensation were assessed using a visual analog scale. The subjective level of fatigue increased and that of appetite for fatty foods showed a tendency toward increase in the target task while the subjective level of fatigue and that of appetite for fatty foods were not altered in the control task. In the target task, the decrease of theta (4-8 Hz) band power in the supplementary motor area (SMA), which was observed in the control task, was suppressed, and the suppression was positively correlated with appetite for fatty foods, suggesting hand grip activity-induced fatigue sensation may increase the appetite for fatty food; this increase could be related to neural activity in the SMA. These findings are expected to contribute to the understanding of the neural mechanisms of appetite in relation to fatigue.

Decreased alpha-band oscillatory brain activity prior to movement initiated by perception of fatigue sensation.

Fatigue is a health problem prevalent in modern societies. Fatigue sensation plays an important role as a biological alarm urging rest to maintain homeostasis, and clarifying the neural mechanisms related to fatigue sensations by which we decide to engage in rest is therefore essential. This study enrolled healthy male volunteers and showed that the decrease in alpha-band power as assessed by magnetoencephalography of the left Brodmann's area (BA) 6 before perception of fatigue when a button-press based on the level of fatigue was required was smaller than that before perception of the intention to move when a voluntary button-press was required. In addition, the decrease of alpha-band power in the left BA 6 before the perception of fatigue was not altered compared with that in the right BA 6 when a button-press based on the level of fatigue was required. These results suggest that the button-press based on the perception of fatigue is not prepared before the perception of fatigue. These findings will advance the understanding of the neural mechanisms related to subjective feelings such as fatigue sensation.

Neural activity induced by visual food stimuli presented out of awareness: a preliminary magnetoencephalography study.

Obesity is a major public health problem in modern society. Appetitive behavior has been proposed to be partially driven by unconscious decision-making processes and thus, targeting the unconscious cognitive processes related to eating behavior is essential to develop strategies for overweight individuals and obese patients. Here, we presented food pictures below the threshold of awareness to healthy male volunteers and examined neural activity related to appetitive behavior using magnetoencephalography. We found that, among participants who did not recognize food pictures during the experiment, an index of heart rate variability assessed by electrocardiography (low-frequency component power/high-frequency component power ratio, LF/HF) just after picture presentation was increased compared with that just before presentation, and the increase in LF/HF was negatively associated with the score for cognitive restraint of food intake. In addition, increased LF/HF was negatively associated with increased alpha band power in Brodmann area (BA) 47 caused by food pictures presented below the threshold of awareness, and level of cognitive restraint was positively associated with increased alpha band power in BA13. Our findings may provide valuable clues to the development of methods assessing unconscious regulation of appetite and offer avenues for further study of the neural mechanisms related to eating behavior.

Immunochemical quantification of cynomolgus CYP2J2, CYP4A and CYP4F enzymes in liver and small intestine.

1. An increasing number of studies have indicated the roles of CYP4 proteins in drug metabolism; however, CYP4 expression has not been measured in cynomolgus monkeys, an important animal species for drug metabolism studies. 2. In this study, cynomolgus CYP4A11, CYP4F2/3, CYP4F11 and CYP4F12, along with CYP2J2, were immunoquantified using selective antibodies in 28 livers and 35 small intestines, and their content was compared with CYP1A, CYP2A, CYP2B6, CYP2C9/19, CYP2D, CYP2E1, CYP3A4 and CYP3A5, previously quantified. 3. In livers, CYP2J2, CYP4A11, CYP4F2/3, CYP4F11 and CYP4F12, varied 1.3- to 4.3-fold, represented 11.2, 14.4, 8.0, 2.7 and 0.3% of total immunoquantified CYP1-4 proteins, respectively. 4. In small intestines, CYP2J2, CYP4F2/3, CYP4F11 and CYP4F12, varied 2.4- to 9.7-fold, represented 6.9, 36.4, 2.4 and 9.3% of total immunoquantified CYP1-4 proteins, respectively, making CYP4F the most abundant P450 subfamily in small intestines. CYP4A11 was under the detection limit in all of the samples analyzed. 5. Significant correlations were found in liver for CYP4A11 with lauric acid 11-/12-hydroxylation and for CYP4F2/3 and CYP4F11 with astemizole hydroxylation. 6. This study revealed the relatively abundant contents of cynomolgus CYP2J2, CYP4A11 and CYP4Fs in liver and/or small intestine, suggesting their potential roles for the metabolism of xenobitotics and endogenous substrates.

Immunochemical detection of cytochrome P450 enzymes in small intestine microsomes of male and female untreated juvenile cynomolgus monkeys.

The expression of small intestinal cytochromes P450 (P450s) has not been systematically measured in cynomolgus monkeys, which are widely used in preclinical drug studies to predict pharmacokinetics and toxicity in humans: therefore, P450 content of small intestine was quantified in 35 cynomolgus monkeys by immunoblotting using 11 selective antibodies. CYP2D, CYP2J2, CYP3A4 and CYP3A5 were detected in all 35 animals, while CYP1A and CYP2C9/19 were detected in 31 and 17 animals, respectively. CYP2C9 and CYP2C19 were detected with the same antibody. CYP1D, CYP2A, CYP2B6, CYP2C76 and CYP2E1 were not detected in any of the 35 animals examined. On analysis of pooled microsomes (35 animals), CYP3A (3A4+3A5) was most abundant (79% of total immunoquantified CYP1-3 proteins), followed by CYP2J2 (13%), CYP2C9/19 (4%), CYP1A (3%) and CYP2D (0.4%). On the analysis of individual microsome samples, each P450 content varied 2-to-6-fold between animals, and no sex differences were observed in any P450 content. These findings should help to increase the understanding of drug metabolism, especially the first-pass effect, in cynomolgus monkey small intestines.

Evaluation of 23 lots of commercially available cryopreserved hepatocytes for induction assays of human cytochromes P450.

Due to the importance of in vitro cytochrome P450 (P450) induction assay to assess the possible drug-drug interaction events, the recent US Food and Drug Administration draft guidance and European Medicines Agency guideline recommend to assess P450 induction using fresh or cryopreserved hepatocytes at mRNA level and/or enzyme activity level. Although cryopreserved hepatocytes are commercially available for P450 induction assays, feasibility and practicability of these hepatocytes have not been fully investigated. In this study, a total of 23 lots of human cryopreserved hepatocytes were treated with three typical inducers (omeprazole, phenobarbital, and rifampicin), and induction of CYP1A2, CYP2B6, and CYP3A4 enzyme activity was measured. In 8 of these 23 hepatocyte lots, induction of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4 mRNA was also analyzed. The results revealed that CYP1A2, CYP2B6, and CYP3A4 were induced (>2.0-fold) by omeprazole, phenobarbital, and rifampicin, respectively, in all the hepatocyte lots tested at enzyme activity level (23 lots) and mRNA level (8 lots). In contrast, of the 8 hepatocyte lots treated with rifampicin, CYP2C8 and CYP2C9 mRNA were not induced in 5 and 2 hepatocyte lots, respectively, and CYP2C19 mRNA was not induced in any of the 8 hepatocyte lots tested. These results suggest that induction of CYP1A2, CYP2B6, and CYP3A4 can be readily assessed, but evaluation for CYP2C mRNA induction might not be feasible, using commercially available human cryopreserved hepatocytes.

Cytochrome P450 metabolic activities in the small intestine of cynomolgus macaques bred in Cambodia, China, and Indonesia.

Cynomolgus macaques, used in drug metabolism studies due to their evolutionary closeness to humans, are mainly bred in Asian countries, including Cambodia, China, and Indonesia. Cytochromes P450 (P450s) are important drug-metabolizing enzymes, present in the liver and small intestine, major drug metabolizing organs. Previously, our investigation did not find statistically significant differences in hepatic P450 metabolic activities measured in cynomolgus macaques bred in Cambodia (MacfaCAM) and China (MacfaCHN). In the present study, P450 metabolic activity was investigated in the small intestine of MacfaCAM and MacfaCHN, and cynomolgus macaques bred in Indonesia (MacfaIDN) using P450 substrates, including 7-ethoxyresorufin, coumarin, bupropion, paclitaxel, diclofenac, S-mephenytoin, bufuralol, chlorzoxazone, and testosterone. The results indicated that P450 metabolic activity of the small intestine was not statistically significantly different (<2.0-fold) in MacfaCAM, MacfaCHN, and MacfaIDN. In addition, statistically significant sex differences were not observed (<2.0-fold) in any P450 metabolic activity in MacfaCAM as supported by mRNA expression results. These results suggest that P450 metabolic activity of the small intestine does not significantly differ statistically among MacfaCAM, MacfaCHN, and MacfaIDN.

Expression profile of hepatic genes in cynomolgus macaques bred in Cambodia, China, and Indonesia: implications for cytochrome P450 genes.

Cynomolgus macaques, frequently used in drug metabolism studies, are bred mainly in the countries of Asia; however, comparative studies of drug metabolism between cynomolgus macaques bred in these countries have not been conducted. In this study, hepatic gene expression profiles of cynomolgus macaques bred in Cambodia (mfCAM), China (mfCHN), and Indonesia (mfIDN) were analyzed. Microarray analysis revealed that expression of most hepatic genes, including drug-metabolizing enzyme genes, was not substantially different between mfCAM, mfCHN, and mfIDN; only 1.1% and 3.0% of all the gene probes detected differential expression (>2.5-fold) in mfCAM compared with mfCHN and mfIDN, respectively. Quantitative polymerase chain reaction showed that the expression levels of 14 cytochromes P450 (P450s) important for drug metabolism did not differ (>2.5-fold) in mfCAM, mfCHN, and mfIDN, validating the microarray data. In contrast, expression of CYP2B6 and CYP3A4 differed (>2.5-fold, p < 0.05) between cynomolgus (mfCAM, mfCHN, or mfIDN) and rhesus macaques, indicating greater differences in expression of P450 genes between the two lineages. Moreover, metabolic activities measured using 14 P450 substrates did not differ substantially (<1.5-fold) between mfCAM and mfCHN. These results suggest that gene expression profiles, including drug-metabolizing enzyme genes such as P450 genes, are similar in mfCAM, mfCHN, and mfIDN.

IL-27 suppresses RANKL expression in CD4+ T cells in part through STAT3.

The receptor activator of NF-κB ligand (RANKL), which is expressed by not only osteoblasts but also activated T cells, plays an important role in bone-destructive diseases such as rheumatoid arthritis. IL-27, a member of the IL-6/IL-12 family cytokines, activates STAT1 and STAT3, promotes early helper T (Th)1 differentiation and generation of IL-10-producing type 1 regulatory T (Tr1) cells, and suppresses the production of inflammatory cytokines and inhibits Th2 differentiation. In addition, IL-27 was recently demonstrated to not only inhibit Th17 differentiation but also directly act on osteoclast precursor cells and suppress RANKL-mediated osteoclastogenesis through STAT1-dependent inhibition of c-Fos, leading to amelioration of the inflammatory bone destruction. In the present study, we investigated the effect of IL-27 on the expression of RANKL in CD4(+) T cells. We found that IL-27 greatly inhibits cell surface expression of RANKL on naive CD4(+) T cells activated by T cell receptor ligation and secretion of its soluble RANKL as well. The inhibitory effect was mediated in part by STAT3 but not by STAT1 or IL-10. In contrast, in differentiated Th17 cells, IL-27 much less efficiently inhibited the RANKL expression after restimulation. Taken together, these results indicate that IL-27 greatly inhibits primary RANKL expression in CD4(+) T cells, which could contribute to the suppressive effects of IL-27 on the inflammatory bone destruction.

Regional distribution of drug-metabolizing enzyme activities in the liver and small intestine of cynomolgus monkeys.

The cynomolgus monkey is an animal species widely used to study drug metabolism because of its evolutionary closeness to humans. However, drug-metabolizing enzyme activities have not been compared in various parts of the liver and small intestine in cynomolgus monkeys. In this study, therefore, drug-metabolizing enzyme activities were analyzed in the liver (the five lobes) and small intestine (six sections from the duodenum to the distal ileum). 7-Ethoxyresorufin O-deethylation, coumarin 7-hydroxylation, paclitaxel 6α-hydroxylation, diclofenac 4'-hydroxylation, tolbutamide methylhydroxylation, S-mephenytoin 4'-hydroxylation, bufuralol 1'-hydroxylation, chlorzoxazone 6-hydroxylation, midazolam 1'-hydroxylation, and testosterone 6ß-, 16α-, 16ß-, and 2α-hydroxylation were used as the probe reactions for this investigation. In liver, all probe reactions were detected and enzyme activity levels were similar in all lobes, whereas, in the small intestine, all enzyme activities were detected (except for coumarin 7-hydroxylase and testosterone 16α-hydroxylase activity), but from jejunum to ileum there was a decrease in the level of enzyme activity. This includes midazolam 1'-hydroxylation and testosterone 6ß-hydroxylation, which are catalyzed by cynomolgus monkey cytochrome P450 (CYP) 3A4/5, orthologs of human CYP3A4/5, which are important drug-metabolizing enzymes. The data presented in this study are expected to facilitate the use of cynomolgus monkeys in drug metabolism studies.

Cynomolgus macaque CYP4 isoforms are functional, metabolizing arachidonic acid.

Cytochrome P450 (CYP) is important for metabolism of not only xenobiotics such as drugs, but also endogenous compounds including arachidonic acids. CYP4A11, CYP4F3v2, CYP4F11, and CYP4F45 have been identified in cynomolgus macaque, an animal species widely used for investigation of drug metabolism due to its evolutionary closeness to human. However, their metabolic functions have not been investigated. In this study, proteins were heterologously expressed in Escherichia coli and characterized by metabolic assays using arachidonic acids as substrates that are metabolized by CYP4 isoforms in human. The results showed that all four CYPs metabolized arachidonic acids. Therefore, cynomolgus macaque CYP4A11, CYP4F3v2, CYP4F11, and CYP4F45 are functional enzymes.

Regional distribution of cytochrome p450 mRNA expression in the liver and small intestine of cynomolgus monkeys.

The cynomolgus monkey is used to study drug metabolism because of its evolutionary closeness to humans. Despite their importance, regional distribution of cytochrome P450 (CYP) enzymes including CYP3As in the liver and small intestine, the major sites of drug metabolism, has not been fully investigated in cynomolgus monkeys. In this study, we measured mRNA expression levels of 14 CYPs in the CYP1, 2, and 3 subfamilies, including orthologs of human CYP3A4 and CYP3A5, in the liver and small intestine of cynomolgus monkeys. Expression levels of each CYP mRNA in various regions of the liver were quantified and comparisons were made between the right lobe, quadrate lobe, left medial lobe, left lateral lobe, and caudate lobe and with four different sections of the right lobe. In the small intestine, the same mRNAs were measured in the duodenum and six different sections from the proximal jejunum to the distal ileum. Expression levels of the CYP mRNAs were not substantially different between liver samples, but varied between the different sections of the small intestine, including CYP3A4. These results suggest that analysis of distinct sections is required for a better understanding of cynomolgus monkey CYPs in the small intestine.

Identification and characterization of CYP2C18 in the cynomolgus macaque (Macaca fascicularis).

The macaque is widely used for investigation of drug metabolism due to its evolutionary closeness to the human. However, the genetic backgrounds of drug-metabolizing enzymes have not been fully investigated; therefore, identification and characterization of drug-metabolizing enzyme genes are important for understanding drug metabolism in this species. In this study, we isolated and characterized a novel cytochrome P450 2C18 (CYP2C18) cDNA in cynomolgus macaques. This cDNA was highly homologous (96%) to human CYP2C18 cDNA. Cynomolgus CYP2C18 was preferentially expressed in the liver and kidney. Moreover, a metabolic assay using cynomolgus CYP2C18 protein heterologously expressed in Escherichia coli revealed its activity toward S-mephenytoin 4'-hydroxylation. These results suggest that cynomolgus CYP2C18 could function as a drug-metabolizing enzyme in the liver.

Effect of a Large Dose of Di (2-ethylhexyl) phthalate (DEHP) on Hepatic Peroxisome in Cynomolgus Monkeys (Macaca Fascicularis).

To elucidate the effect of a large dose of di (2-ethylhexyl) phthalate (DEHP), a plasticizer and peroxisome proliferator-activated receptor-α (PPARα) agonist, on hepatic peroxisomes, we orally administered 1,000 mg/kg/day, once daily, to 3 male and 4 female cynomolgus monkeys for 28 days consecutively. Light-microscopic and electron microscopic examinations of the liver were carried out in conjunction with measurement of the hepatic fatty acid ß-oxidation system (FAOS), carnitine acetyltransferase (CAT) and carnitine palmitoyltransferase (CPT) activities, which are peroxisomal and/or mitochondrial enzyme activities. Electron microscopically, enlargement of the mitochondria was observed with lamellar orientation of the cristae along the major axis. Although the number of peroxisomes showed a tendency to increase when compared with those in a biopsied specimen before treatment, no abnormality in morphology was observed. A slight increase in CPT activity was noted at termination. No changes were noted in hepatic FAOS or CAT activity. In conclusion, although repeated oral treatment of cynomolgus monkeys with a large dose of DEHP induced a subtle increase in the numbers of peroxisomes with slight enlargements of the mitochondria, this low-sensitivity response to peroxisome proliferators in cynomolgus monkeys was considered to be closer to the response in humans than that in rodents.

A practical novel method for ensuring stable capacitation of spermatozoa from cryopreserved C57BL/6J sperm suspension.

A large number of genetically modified mouse strains have been produced in recent years. Sperm cryopreservation is the most effective means of preserving these valuable strains, most of which have a C57BL/6 genetic background. However, the fertilization efficiency of sperm from several cryopreserved strains, including C57BL/6, is quite low. While new and improved methods of cryopreservation have been developed, the majority of sperm stocks have already been cryopreserved using traditional methods, such as storage in 18% raffinose and 3% skim milk (R18S3). Therefore, new thawing methods for these frozen stocks are needed. We have developed a new thawing method that involves selective collection of motile sperm and a preincubation medium that enhances capacitation. Motile sperm are selected simply by collecting a sample from the center of a dish, and capacitation is induced by the addition of methyl-beta-cyclodextrin, D-penicillamine, sodium citrate, and hypotaurine to modified Tyrode's solution. The fertilization rate of sperm prepared using this method was increased significantly compared to that of sperm thawed using the traditional method (63.9 vs 16.5%, P<0.01). These results demonstrate that this new in vitro fertilization method is an effective means of reviving C57BL/6 sperm cryopreserved in R18S3.

Identification and characterization of CYP2B6 cDNA in cynomolgus macaques (Macaca fascicularis).

Cytochrome P450 2B6 (CYP2B6), an important drug-metabolizing enzyme, is involved in the metabolism of prescribed drugs in humans. Despite its importance, cDNA for a CYP2B6 ortholog has not been identified and characterized in cynomolgus macaques, which are frequently used in preclinical studies. In this study, cDNA highly homologous to human CYP2B6 was cloned from the cynomolgus macaque liver. This cDNA contained an open reading frame of 491 amino acids and functional domains characteristic for CYP protein, such as substrate recognition sites and a heme-binding region. Cynomolgus CYP2B6 was expressed predominantly in the liver with some extra-hepatic expression among 10 tissues. Moreover, cynomolgus CYP2B6 revealed activities toward testosterone 16beta-hydroxylation and bupropion hydroxylation. These results suggest that cynomolgus CYP2B6 has a functional role in the liver.

A null allele impairs function of CYP2C76 gene in cynomolgus monkeys: a possible genetic tool for generation of a better animal model in drug metabolism.

The monkey CYP2C76 gene does not correspond to any of the human CYP2C genes, and its enzyme is at least partly responsible for the species difference occasionally seen in drug metabolism between monkeys and humans. To establish a line and/or lines of monkeys that are expected to show metabolic patterns highly similar to humans, we set out to find monkeys that lacked CYP2C76 activity. By genetic screening of 73 monkeys and a database search of expressed sequence tags, we found a total of 10 nonsynonymous genetic variants in the coding region of CYP2C76, including a null genotype (c.449TG>A). Some of the variants were differently distributed between two animal groups originating from different geographical regions (Indochina and Indonesia). After screening 170 additional genomic samples, we identified a total of eight animals (six males and two females) that were heterozygous for c.449TG>A, which could be used for establishing a homozygous line. If the homozygotes show drug-metabolizing properties more similar to humans than wild-type monkeys, the homozygotes may serve as a better animal model for drug metabolism. The data presented in this article provide the essential genetic information to perform a successful study by using cynomolgus monkeys and present a possible tool to generate a better animal model for drug metabolism.