The importance of accounting for testing and positivity in surveillance by time and place: an illustration from HIV surveillance in Japan.

The number of tests performed is an important surveillance indicator. We illustrate this point using HIV surveillance data, focusing on Tokyo and Okinawa, two prefectures with high HIV notification rates in Japan. Restricting to data reported from local public health centres and affiliate centres where testing data are accessible, we assessed HIV surveillance data during 2007-2014, based on the annual HIV notification rate (per 100 000 population), HIV testing rate (per 100 000 population) and proportion testing HIV-positive (positivity). Nationally, testing activity and positivity showed an inverse relationship; in 2008, the testing rate peaked, but positivity was lowest. While notification rates were higher for Tokyo (median = 0.98, range = 0.89-1.33) than Okinawa (median = 0.61, range = 0.42-1.09), Okinawa had slightly higher testing rates (median = 187, range = 158-274) relative to Tokyo (median = 172, range = 163-210). Positivity was substantially lower in Okinawa (median = 0.34%, range = 0.24-0.45%) compared with Tokyo (median = 0.57%, range = 0.46-0.67%). Relative to the national testing rate (median = 85, range = 80-115) and positivity (median = 0.34%, range = 0.28-0.36%), Tokyo had higher positivity, despite more testing. In 2014 in Okinawa, all three indicators increased, providing a strong reason to be concerned as positivity increased despite more testing. Together with other information, accounting for testing and positivity improve interpretation of surveillance data to guide public health assessments.

Local outbreak of Streptococcus pneumoniae serotype 12F caused high morbidity and mortality among children and adults.

Pneumococcal serotype replacement is an important issue after the introduction of pneumococcal conjugate vaccine (PCV) in children. After the introduction of 13-valent PCV, the incidence of invasive pneumococcal diseases (IPD) caused by Streptococcus pneumoniae serotype 12F (Sp12F) have increased in some countries; however, an outbreak of Sp12F has not reported in the post-13-valent PCV era. We experienced a local outbreak of Sp12F during March through May 2016 in Tsuruoka city, Japan after the introduction of 13-valent PCV in 2013. The IPD patients were two children and seven adults, three of whom died with a rapid disease progress. Although the clear transmission route was not determined, eight of the nine patients (89%) had close contact with children, which suggests that transmitted colonisation of Sp12F among children and adults might be the source of transmission. Continuous monitoring of IPDs, along with the determination of pneumococcal serotypes, is warranted in the post-13-valent PCV era. New IPD control strategies may be needed if this fatal outbreak continues to occur.

HRCT findings of collagen vascular disease-related interstitial pneumonia (CVD-IP): a comparative study among individual underlying diseases.

AIM: To identify characteristic high-resolution computed tomography (CT) findings for individual collagen vascular disease (CVD)-related interstitial pneumonias (IPs). MATERIALS AND METHODS: The HRCT findings of 187 patients with CVD, including 55 patients with rheumatoid arthritis (RA), 50 with systemic sclerosis (SSc), 46 with polymyositis/dermatomyositis (PM/DM), 15 with mixed connective tissue disease, 11 with primary Sjögren's syndrome, and 10 with systemic lupus erythematosus, were evaluated. Lung parenchymal abnormalities were compared among CVDs using χ2 test, Kruskal-Wallis test, and multiple logistic regression analysis. A CT-pathology correlation was performed in 23 patients. RESULTS: In RA-IP, honeycombing was identified as the significant indicator based on multiple logistic regression analyses. Traction bronchiectasis (81.8%) was further identified as the most frequent finding based on χ2 test. In SSc IP, lymph node enlargement and oesophageal dilatation were identified as the indicators based on multiple logistic regression analyses, and ground-glass opacity (GGO) was the most extensive based on Kruskal-Wallis test, which reflects the higher frequency of the pathological nonspecific interstitial pneumonia (NSIP) pattern present in the CT-pathology correlation. In PM/DM IP, airspace consolidation and the absence of honeycombing were identified as the indicators based on multiple logistic regression analyses, and predominance of consolidation over GGO (32.6%) and predominant subpleural distribution of GGO/consolidation (41.3%) were further identified as the most frequent findings based on χ2 test, which reflects the higher frequency of the pathological NSIP and/or the organising pneumonia patterns present in the CT-pathology correlation. CONCLUSION: Several characteristic high-resolution CT findings with utility for estimating underlying CVD were identified.

Association of immunoglobulin G4 and free light chain with idiopathic pleural effusion.

The cause of pleural effusion remains uncertain in approximately 15% of patients despite exhaustive evaluation. As recently described immunoglobulin (Ig)G4-related disease is a fibroinflammatory disorder that can affect various organs, including the lungs, we investigate whether idiopathic pleural effusion includes IgG4-associated etiology. Between 2000 and 2012, we collected 830 pleural fluid samples and reviewed 35 patients with pleural effusions undiagnosed after pleural biopsy at Yamaguchi-Ube Medical Center. Importantly, IgG4 immunostaining revealed infiltration of IgG4-positive plasma cells in the pleura of 12 patients (34%, IgG4+ group). The median effusion IgG4 level was 41 mg/dl in the IgG4+ group and 27 mg/dl in the IgG4- group (P < 0·01). The light and heavy chains of effusion IgG4 antibodies of patients in the IgG4+ group were heterogeneous by two-dimensional electrophoresis, indicating the absence of clonality of the IgG4 antibodies. Interestingly, the κ light chains were more heterogeneous than the λ light chains. The measurement of the κ and λ free light chain (FLC) levels in the pleural fluids showed significantly different κ FLC levels (median: 28·0 versus 9·1 mg/dl, P < 0·01) and κ/λ ratios (median: 2·0 versus 1·2, P < 0·001) between the IgG4+ and IgG4- groups. Furthermore, the κ/λ ratios were correlated with the IgG4+ /IgG+ plasma cell ratios in the pleura of the IgG4+ group. Taken together, these results demonstrate the involvement of IgG4 in certain idiopathic pleural effusions and provide insights into the diagnosis, pathogenesis and therapeutic opportunities of IgG4-associated pleural effusion.

Comprehensive population screening in the Ashkenazi Jewish population for recurrent disease-causing variants.

The Ashkenazi Jewish (AJ) population has an increased risk for a variety of recessive diseases due to historical founder effects and genetic drift. For some, the disease-causing founder mutations have been identified and well-characterized, but for others, further study is necessary. The purpose of this study is to assess the carrier frequencies of 85 pathogenic variants causative of 29 recessive conditions in the AJ population. Up to 3000 AJ individuals were genotyped by Luminex MagPlex® -TAG™ bead array or Agena Bioscience™ MassARRAY assays. We identified seven conditions with carrier frequencies higher than 1 in 100, nine between 1 in 100 and 1 in 200, and four between 1 in 200 and 1 in 500. Variants in nine conditions had a detected carrier rate of less than 1 in 500 or were not identified in approximately 2000 AJ individuals. We assessed the combined AJ carrier frequency for 18 relatively prevalent diseases to be 1 in 6, and the risk of AJ individuals to be a carrier couple for one of these 18 diseases as 1 in 441. We note additional recessive genetic conditions should be considered for AJ carrier screening panels.

Increase in paratyphoid fever cases in Japanese travellers returning from Cambodia in 2013.

In 2013, an unusual increase of paratyphoid fever cases in travellers returning from Cambodia was reported in Japan. From December 2012 to September 2013, 18 cases of Salmonella Paratyphi A infection were identified. Microbiological analyses revealed that most isolates had the same clonal identity, although the epidemiological link between these cases remains unclear. It was inferred that the outbreak was caused by a common and persistent source in Cambodia that was likely to have continued during 2014. The information of surveillance and laboratory data from cases arising in travellers from countries with limited surveillance systems should be timely shared with the country of origin.

ITPKC and CASP3 polymorphisms and risks for IVIG unresponsiveness and coronary artery lesion formation in Kawasaki disease.

Functional single-nucleotide polymorphisms (SNPs) in inositol 1,4,5-trisphosphate 3-kinase C (ITPKC) (rs28493229) and caspase-3 (CASP3) (rs113420705; formerly rs72689236) are associated with susceptibility to Kawasaki's disease (KD). To evaluate the involvement of these 2 SNPs in the risk for intravenous immunoglobulin (IVIG) unresponsiveness, we investigated 204 Japanese KD patients who received a single IVIG dose of 2 g kg(-1) (n=70) or 1 g kg(-1) daily for 2 days (n=134). The susceptibility allele of both SNPs showed a trend of overrepresentation in IVIG non-responders and, in combined analysis of these SNPs, patients with at least 1 susceptible allele at both loci had a higher risk for IVIG unresponsiveness (P=0.0014). In 335 prospectively collected KD patients who were treated with IVIG (2 g kg(-1)), this 2-locus model showed a more significant association with resistance to initial and additional IVIG (P=0.011) compared with individual SNPs. We observed a significant association when all KD patients with coronary artery lesions were analyzed with the 2-locus model (P=0.0031). Our findings strongly suggest the existence of genetic factors affecting patients' responses to treatment and the risk for cardiac complications, and provide clues toward understanding the pathophysiology of KD inflammation.

Alcohol consumption promotes the intestinal translocation of Streptococcus suis infections.

Streptococcus suis is an emerging zoonotic agent. This study aimed to investigate whether S. suis is likely to translocate across the intestines of human hosts who have liver disease and/or consume alcohol. Both the alcoholism and cirrhosis models exhibited high mRNA expression of TGF and collagen1, but only the cirrhosis model had fibrosis in the liver. After both models were infected with S. suis, significantly different concentrations of S. suis were detected in the blood and brains of the alcoholism model (Blood: 36.4%; Brain: 31.8%) and the cirrhosis model (Blood: 62.5%; Brain: 62.5%) compared to the concentrations in the healthy mice (Blood: 15.4%; Brain: 0%). Trans-epithelial electrical resistance (TER) was used to examine the Caco-2 cells in the in vitro that had an S. suis infection combined with 1% ethanol. Although the ethanol did not influence the Caco-2 cells' barriers, it did rapidly decrease the barriers' TER value and then their E-cadherin compared to the infected Caco-2 cells without the ethanol treatment. Immunofluorescence also indicated that the barriers of the Caco-2 cells treated with ethanol were disrupted and that S. suis translocated from the apical to the basolateral side. This study demonstrated that alcohol consumption helped S. suis to translocate.

Mutations in a new gene encoding a thiamine transporter cause thiamine-responsive megaloblastic anaemia syndrome.

Thiamine-responsive megaloblastic anaemia syndrome (TRMA; MIM 249270) is an autosomal recessive disorder with features that include megaloblastic anaemia, mild thrombocytopenia and leucopenia, sensorineural deafness and diabetes mellitus. Treatment with pharmacologic doses of thiamine ameliorates the megaloblastic anaemia and diabetes mellitus. A defect in the plasma membrane transport of thiamine has been demonstrated in erythrocytes and cultured skin fibroblasts from TRMA patients. The gene causing TRMA was assigned to 1q23.2-q23.3 by linkage analysis. Here we report the cloning of a new gene, SLC19A2, identified from high-through-put genomic sequences due to homology with SLC19A1, encoding reduced folate carrier 1 (refs 8-10). We cloned the entire coding region by screening a human fetal brain cDNA library. SLC19A2 encodes a protein (of 497 aa) predicted to have 12 transmembrane domains. We identified 2 frameshift mutations in exon 2. a 1-bp insertion and a 2-bp deletion, among four Iranian families with TRMA. The sequence homology and predicted structure of SLC19A2, as well as its role in TRMA, suggest that its gene product is a thiamine carrier, the first to be identified in complex eukaryotes.

Effects of liquefied sake lees on growth performance and faecal and blood characteristics in Japanese Black calves.

Liquefied sake lees, a by-product of Japanese sake, is rich in Saccharomyces cerevisiae, proteins, and prebiotics derived from rice and yeast. Previous studies have reported that Saccharomyces cerevisiae fermentation products improved the health, growth, and faecal characteristics of preweaning calves. This study investigated the effects of adding liquefied sake lees to milk replacer on the growth performance, faecal characteristics, and blood metabolites of preweaning Japanese Black calves from 6 to 90 days of age. Twenty-four Japanese Black calves at 6 days of age were randomly assigned to one of three treatments: No liquefied sake lees (C, n = 8), 100 g/d (on a fresh matter basis) liquefied sake lees mixed with milk replacer (LS, n = 8), and 200 g/d (on a fresh matter basis) liquefied sake lees mixed with milk replacer (HS, n = 8). The intake of milk replacer and calf starter, as well as, the average daily gain did not differ between the treatments. The number of days counted with faecal score 1 in LS was higher than in HS (P < 0.05), while the number of days with diarrhoea medication in LS and C was lower than HS (P < 0.05). The faecal n-butyric acid concentration tended to be higher in LS compared to C (P = 0.060). The alpha diversity index (Chao1) was higher in HS than in C and LS at 90 days of age (P < 0.05). The principal coordinate analysis (PCoA) using weighted UniFrac distance showed that the bacterial community structures in faeces among the treatments at 90 days of age were significantly different (P < 0.05). The plasma ß-hydroxybutyric acid concentration, an indicator of rumen development, was higher for LS than in C throughout the experiment (P < 0.05). These results suggested that adding liquefied sake lees up to 100 g/d (on a fresh matter basis) might promote rumen development in preweaning Japanese Black calves.

Effectiveness of patient and staff cohorting to reduce the risk of vancomycin-resistant enterococcus (VRE) acquisition: a retrospective cohort study during a VRE outbreak in Japan.

BACKGROUND: Patient and staff cohorting is part of a bundle approach in the response to multi-drug-resistant organisms, but its effectiveness is not fully clarified. This study compared the risks of acquiring vancomycin-resistant Enterococcus faecium (VREfm) at a hospital during a VREfm outbreak based on contact characteristics in order to better understand the effectiveness of cohorting. METHODS: Exposure came from contact with patients with VREfm (infectors), including existing patients with VREfm and patients who acquired VREfm during the study period. Contact was defined as length of contact time, degree of sharing space, and care by the same nurses as those caring for infectors between January and March 2018. The outcome was VREfm acquisition as determined through monthly stool or rectal screening cultures. Incidence rates were calculated based on contact patterns, and incidence rate ratios (IRRs) were compared. FINDINGS: Among 272 inpatients (4038 patient-days), 43 patients acquired VREfm with the same or similar pulsotype. Incidence rates were 8.45 per 1000 patient-days when susceptible inpatients were on the same ward as an infector but cared for by different nurses (reference), 16.96 when susceptible inpatients were on the same ward as an infector and cared for by the same nurses [IRR 2.01, 95% confidence interval (CI) 0.62-10.28], and 52.91 when susceptible inpatients shared a room with an infector (IRR 6.26, 95% CI 1.61-35.40). CONCLUSION: Compared with susceptible inpatients in a different room from infectors and not being cared for by the same nurses, the risk of VREfm acquisition could be six times higher for susceptible inpatients who are in the same room as infectors, and could be double for susceptible inpatients cared for by the same nurses as infectors.

ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons.

The ERM family members, ezrin, radixin, and moesin, localizing just beneath the plasma membranes, are thought to be involved in the actin filament/plasma membrane association. To identify the integral membrane protein directly associated with ERM family members, we performed immunoprecipitation studies using antimoesin mAb and cultured baby hamster kidney (BHK) cells metabolically labeled with [35S]methionine or surface-labeled with biotin. The results indicated that moesin is directly associated with a 140-kD integral membrane protein. Using BHK cells as antigens, we obtained a mAb that recognized the 140-kD membrane protein. We next cloned a cDNA encoding the 140-kD membrane protein and identified it as CD44, a broadly distributed cell surface glycoprotein. Immunoprecipitation with various anti-CD44 mAbs showed that ezrin and radixin, as well as moesin, are associated with CD44, not only in BHK cells, but also in mouse L fibroblasts. Furthermore, immunofluorescence microscopy revealed that in both BHK and L cells, the Triton X-100-insoluble CD44 is precisely colocalized with ERM family members. We concluded that ERM family members work as molecular linkers between the cytoplasmic domain of CD44 and actin-based cytoskeletons.

Specific proto-oncogenic tyrosine kinases of src family are enriched in cell-to-cell adherens junctions where the level of tyrosine phosphorylation is elevated.

To approach the transmembrane signaling pathway in the cell-to-cell adherens junctions (AJ), AJ-specific tyrosine phosphorylation was analyzed. When various types of rat adult tissues were pretreated with sodium orthovanadate, a potent inhibitor of tyrosine phosphatase, immunofluorescence microscopy showed that anti-phosphotyrosine polyclonal antibody specifically stained the undercoat of the cell-to-cell AJ. This indicates that the tyrosine kinase activity is elevated at the undercoat of the cell-to-cell AJ of adult tissues. To identify tyrosine kinases responsible for the high level of tyrosine phosphorylation at AJ, we have performed in vitro phosphorylation experiments with cell-to-cell AJ isolated from rat liver (Tsukita, Sh. and Sa. Tsukita. 1989. J. Cell Biol. 108:31-41) and immunoblotting analyses with specific antibodies for tyrosine kinases. As a result, three proto-oncogenic tyrosine kinases of src family, c-yes, c-src, and lyn kinases, were identified as major tyrosine kinases in the cell-to-cell AJ of hepatocytes. Furthermore, it was immunofluorescently shown that at least two of these kinases, c-yes and c-src kinases, were enriched at the cell-to-cell AJ of various types of cells including hepatocytes. Based on these findings, it is concluded that, in various types of cells, specific proto-oncogenic tyrosine kinases of src-family (c-yes and c-src) are enriched to work as signal mediators in the cell-to-cell AJ where the level of tyrosine phosphorylation is elevated.

In vitro development of gut-like tissue demonstrating rhythmic contractions from embryonic mouse intestinal cells.

The rhythmic motility of the intestine is regulated by the interstitial cells of Cajal (ICC) and the enteric nervous system. Rhythmic motility is considered to occur after the differentiation of mesenchymal progenitor cells into ICC during the late embryonic period. In this study, we successfully reconstructed a gut-like tissue demonstrating rhythmic contractions by culturing dispersed cells enzymatically isolated from the mouse intestine during the mid-embryonic period. These intestinal cells were reconstituted into a collagen gel at high density, made to proliferate considerably, and grew into a gut-like tissue after 1 week of culturing. The reconstituted tissue showed rhythmic contractions and stained positive for the specific marker proteins of neurones and ICC, PGP9.5 and c-Kit. The tissue also demonstrated network formation by developing nerve cells and ICC. Moreover, in the presence of nifedipine, c-Kit-immunopositive cells showed spontaneous Ca(2+) oscillation, which is considered to be coupled to the electrical activity that corresponds to slow waves. Therefore, this culture system may be of use in elucidating the developmental mechanisms of gastrointestinal motility.

Characterization of the gene and mRNA of the large subunit of ribulose-1,5-bisphosphate carboxylase in pea plants.

mRNA coding for the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase was obtained by fractionating chloroplast polysomes on an affinity column, using anti-ribulose-1,5-bisphosphate carboxylase immunoglobulin G. Approximately 20% of the polysomal RNA specifically bound to the affinity column. LS mRNA was also isolated by fractionating chloroplast polysomal RNA on sucrose gradients. The LS mRNA fraction was identified by translation in vitro followed by immunoprecipitation with anti-ribulose-1,5-bisphosphate carboxylase immunoglobulin G. Labeled LS mRNA was hybridized to a genomic digests of pea chloroplast DNA. The LS gene was localized on a 3.55-kilobase pair BamHI fragment in SalI-SmaI DNA fragment 4. The BamHI fragment containing the LS gene was cloned, and a restriction endonuclease map was constructed. The LS gene was localized on a 1.9-kbp KpnI-EcoRI fragment. The LS gene was analyzed by electron microscopy, using the R loop mapping technique. LS mRNA was colinear with the gene, and its size was 1.35 +/- 0.2 kilobase pairs. When the LS mRNA was analyzed on methylmercury agarose gels, it comigrated with the 16S rRNA. The direction of transcription of the LS gene was in the same direction as that of the rRNA genes.

Sequence organization of a pea chloroplast DNA gene coding for a 34,500-dalton protein.

A gene (PGII), which codes for a 34.5-kilodalton protein, has been isolated and cloned from pea chloroplast DNA. The production of its 1.2-kilobase mRNA is photodependent. The direction of transcription has been determined, the site of initiation of transcription has been found, and an in vitro protein product has been produced. The gene, including the 5' and 3'-flanking regions, has been sequenced. It shows ca. 95% homology to the photosystem II thylakoid membrane protein, photogene 32, from spinach and tobacco. There are no intervening sequences. The 5'-flanking region suggests similarities with Escherichia coli promoters. The 5'-flanking region is remarkably conserved among pea, spinach, and tobacco DNA.

Estimation of the energy expenditure of grazing ruminants by incorporating dynamic body acceleration into a conventional energy requirement system.

The estimation of energy expenditure (EE) of grazing animals is of great importance for efficient animal management on pasture. In the present study, a method is proposed to estimate EE in grazing animals based on measurements of body acceleration of animals in combination with the conventional Agricultural and Food Research Council (AFRC) energy requirement system. Three-dimensional body acceleration and heart rate were recorded for tested animals under both grazing and housing management. An acceleration index, vectorial dynamic body acceleration (VeDBA), was used to calculate activity allowance (AC) during grazing and then incorporate it into the AFRC system to estimate the EE (EE derived from VeDBA [EE]) of the grazing animals. The method was applied to 3 farm ruminant species (7 cattle, 6 goats, and 4 sheep). Energy expenditure based on heart rate (EE) was also estimated as a reference. The result showed that larger VeDBA and heart rate values were obtained under grazing management, resulting in greater EE and EE under grazing management than under housing management. There were large differences between the EE estimated from the 2 methods, where EE values were greater than EE (averages of 163.4 and 142.5% for housing and grazing management, respectively); the EE was lower than the EE, whereas the increase in EE under grazing in comparison with housing conditions was larger than that in EE. These differences may have been due to the use of an equation for estimating EE derived under laboratory conditions and due to the presence of the effects of physiological, psychological, and environmental factors in addition to physical activity being included in measurements for the heart rate method. The present method allowed us to separate activity-specific EE (i.e., AC) from overall EE, and, in fact, AC under grazing management were about twice times as large as those under housing management for farm ruminant animals. There is evidence that the conventional energy system can predict fasting metabolism and the AC of housed animals based on accumulated research on energy metabolism and that VeDBA can quantify physical activity separately from other factors in animals on pasture. Therefore, the use of the VeDBA appears to be a precise way to predict activity-specific EE under grazing conditions, and the method incorporating acceleration index data with a conventional energy system can be a simple and useful method for estimation of EE in farm ruminants on pastures.

Adrenal gland-dependent augmentation of plasminogen activator inhibitor-1 expression in streptozotocin-induced diabetic mice.

BACKGROUND: Diabetes is associated with an excess risk of cardiac events, and one risk factor for infarction is an elevated level of plasminogen activator inhibitor-1 (PAI-1). OBJECTIVES AND METHODS: To evaluate whether the glucocorticoid hormones are involved in the diabetes-induced PAI-1 production, we examined expression profiles of PAI-1 mRNA in adrenalectomized (ADX) mice with streptozotocin (STZ)-induced diabetes. RESULTS: The diabetes-induced augmentation of plasma PAI-1 levels and PAI-1 mRNA expression in the heart and lungs was completely normalized in diabetic ADX mice. The glucocorticoid receptor antagonist RU486 significantly, but only partly suppressed PAI-1 induction in STZ-induced diabetic mice, suggesting that factors other than glucocorticoids are also involved in PAI-1 induction provoked by diabetes. CONCLUSION: Our results suggested that the adrenal gland plays a critical role in the progression of thrombosis in diabetic patients by inducing expression of the PAI-1 gene.

CLOCK is involved in obesity-induced disordered fibrinolysis in ob/ob mice by regulating PAI-1 gene expression.

BACKGROUND: An increased level of obesity-induced plasma plasminogen activator inhibitor-1 (PAI-1) is considered a risk factor for cardiovascular disease. AIM: The present study investigates whether the circadian clock component CLOCK is involved in obesity-induced PAI-1 elevation. METHODS: We examined plasma PAI-1 and mRNA expression levels in tissues from leptin-deficient obese and diabetic ob/ob mice lacking functional CLOCK protein. RESULTS: Our results demonstrated that plasma PAI-1 levels were augmented in a circadian manner in accordance with the mRNA expression levels in ob/ob mice. Surprisingly, a Clock mutation normalized the plasma PAI-1 concentrations in accordance with the mRNA levels in the heart, lung and liver of ob/ob mice, but significantly increased PAI-1 mRNA levels in adipose tissue by inducing adipocyte hypertrophy in ob/ob mice. The Clock mutation also normalized tissue PAI-1 antigen levels in the liver but not in the adipose tissue of ob/ob mice. CONCLUSION: These observations suggest that CLOCK is involved in obesity-induced disordered fibrinolysis by regulating PAI-1 gene expression in a tissue-dependent manner. Furthermore, it appears that obesity-induced PAI-1 production in adipose tissue is not closely related to systemic PAI-1 increases in vivo.

Circadian clock molecules CLOCK and CRYs modulate fibrinolytic activity by regulating the PAI-1 gene expression.

Disruptions of circadian rhythms are associated with the development of many disorders. However, whether a disruption of the circadian clock can cause anomalies of the hemostatic balance remains unknown. The present study examines coagulation and fibrinolytic activities in circadian clock mutants, a homozygous Clock mutant and Cry1/Cry2 double knockout (Cry1/2-deficient) mice. The euglobulin clot lysis time (ELT) showed circadian variations that peaked at 21:00 (early night) in wild-type mice, suggesting that fibrinolytic activity is lowest at this time. The ELT was continuously reduced in Clock mutants, while the ELT was significantly increased and did not differ between day and night (9:00 and 21:00) in Cry1/2-deficient mice. The prothrombin time (PT) and activated partial prothrombin time (APTT) were constant in all genotypes. To identify which factors cause the loss of ELT rhythm, we measured fibrinolytic parameters in Clock mutant and Cry1/2-deficient mice. The robust circadian fluctuation of plasma plasminogen activator inhibitor 1 (PAI-1) that peaked at early night was damped to trough levels in Clock mutant mice. On the other hand, PAI-1 levels in Cry1/2-deficient mice remained equivalent to the peak levels of those in wild-type mice at both 9:00 and 21:00. Circadian changes in plasma PAI-1 levels seemed to be regulated at the level of gene expression, because the plasma PAI-1 levels in Clock mutant and Cry1/2-deficient mice were closely correlated with the level of PAI-1 mRNA transcript in these mice. Plasma plasminogen and hepatic mRNA levels were not rhythmic in wild-type mice, and continuously higher in Clock mutant than in wild-type or Cry1/2-deficient mice. In contrast, the activity and mRNA levels of tissue type plasminogen activator (t-PA), plasma levels and mRNA levels of plasminogen, and plasma levels of alpha2 plasmin inhibitor (alpha2PI) in all genotypes were constant throughout the day. Coagulation parameters such as factor VII, factor X, prothrombin and fibrinogen remained constant throughout the day, and were not affected by clock gene mutations. These results suggest that circadian clock molecules play an important role in hemostatic balance by regulating the fibrinolytic systems.